Methods and compositions for diagnosing and treating disorders involving angiogenesis

ABSTRACT

The present invention relates to polynucleotides associated with angiogenesis-related disorders. The present invention also relates to canine endostatin genes, novel genes associated with angiogenesis-related disorders, such as cancer. The invention encompasses endostatin nucleic acids, recombinant DNA molecules, cloned genes or degenerate variants thereof, endostatin gene products and antibodies directed against such gene products, cloning vectors containing mammalian endostatin gene molecules, and hosts that have been genetically engineered to express such molecules. The invention further relates to methods for the identification of compounds that modulate the expression of endostatin genes and gene products and to using such compounds as therapeutic agents in the treatment of angiogenesis-related disorders, e.g., cancer. The invention also relates to methods for the diagnostic evaluation, genetic testing and prognosis of angiogenesis-related disorders, e.g., cancer, and to methods and compositions for the treatment these disorders.

INTRODUCTION

The present invention relates to polynucleotide sequences which areshown herein to be associated with the regulation of angiogenesis. Morespecifically, the present invention relates to novel polynucleotidesequences which encode the angiogenesis inhibitor endostatin, and moreparticularly, the canine angiogenesis inhibitor. The inventionencompasses endostatin nucleic acids, recombinant DNA molecules, clonedgenes and degenerate variants thereof, vectors containing suchendostatin nucleic acids, and hosts that have been geneticallyengineered to express and/or contain such molecules. The inventionfurther relates to endostatin gene products and antibodies directedagainst such gene products. The invention further relates to methods forthe identification of compounds that modulate the expression, synthesisand activity of such endostatin nucleic acids, and to methods of usingcompounds such as those identified herein as therapeutic agents in thetreatment of angiogenesis-related disorders, including, but not limitedto, cancer. The invention also relates to methods for the diagnosticevaluation, genetic testing and prognosis of an angiogenesis-relateddisorder, including, but not limited to, cancer.

BACKGROUND OF THE INVENTION

Angiogenesis, defined as the growth or sprouting of new blood vesselsfrom existing vessels, is a complex process that primarily occurs duringembryonic development. Under normal physiological conditions in adults,angiogenesis takes place only in very restricted situations such as hairgrowth and wounding healing (Auerbach, W. and Auerbach, R., 1994,Pharmacol Ther 63(3):265-3 11; Ribatti et al.,1991, Haematologica76(4):3 11-20; Risau, 1997, Nature 386(6626):67 1-4). Unregulatedangiogenesis has gradually been recognized to be responsible for a widerange of disorders, including, but not limited to, cancer,cardiovascular disease, rheumatoid arthritis, psoriasis and diabeticretinopathy (Folkman, 1995, Nat Med 1(1):27-31; Isner, 1999, Circulation99(13): 1653-5; Koch, 1998, Arthritis Rheum 41(6):951-62; Walsh, 1999,Rheumatology (Oxford) 38(2):103-12; Ware and Simons, 1997, Nat Med 3(2):158-64). Of particular interest is the observation that angiogenesis isrequired by solid tumors for their growth and metastases (Folkman, 1986,Cancer Res, 46(2) 467-73. Folkman 1990, J Natl. Cancer Inst., 82(1) 4-6,Folkman, 1992, Semin Cancer Biol 3(2):65-71; Zetter, 1998, Annu Rev Med49:407-24). A tumor usually begins as a single aberrant cell which canproliferate only to a size of a few cubic millimeters due to thedistance from available capillary beds, and it can stay ‘dormant’without further growth and dissemination for a long period of time. Sometumor cells then switch to the angiogenic phenotype to activateendothelial cells, which proliferate and mature into new capillary bloodvessels. These newly formed blood vessels not only allow for continuedgrowth of the primary tumor, but also for the dissemination andrecolonization of metastatic tumor cells. The precise mechanisms thatcontrol the angiogenic switch is not well understood, but it is believedthat neovascularization of tumor mass results from the net balance of amultitude of angiogenesis stimulators and inhibitors (Folkman, 1995, NatMed 1(1):27-31).

One of the most potent angiogenesis inhibitors is endostatin identifiedby O'Reilly and Folkman (O'Reilly et al., 1997, Cell 88(2):277-85;O'Reilly et al., 1994, Cell 79(2):3 15-28). Its discovery was based onthe phenomenon that certain primary tumors can inhibit the growth ofdistant metastases. O'Reilly and Folkman hypothesized that a primarytumor initiates angiogenesis by generating angiogenic stimulators inexcess of inhibitors. However, angiogenic inhibitors, by virtue of theirlonger half life in the circulation, reach the site of a secondary tumorin excess of the stimulators. The net result is the growth of primarytumor and inhibition of secondary tumor. Endostatin is one of a growinglist of such angiogenesis inhibitors produced by primary tumors. It is aproteolytic fragment of a larger protein: endostatin is a 20 kDafragment of collagen XVIII (amino acid H1132-K1315 in murine collagenXVIII). Endostatin has been shown to specifically inhibit endothelialcell proliferation in vitro and block angiogenesis in vivo. Moreimportantly, administration of endostatin to tumor-bearing mice leads tosignificant tumor regression, and no toxicity or drug resistance hasbeen observed even after multiple treatment cycles (Boehm et al., 1997,Nature 390(6658):404-407). The fact that endostatin targets geneticallystable endothelial cells and inhibits a variety of solid tumors makes ita very attractive candidate for anticancer therapy (Fidler and Ellis,1994, Cell 79(2):185-8; Gastl et al., 1997, Oncology 54(3): 177-84; vanHinsbergh et al., 1999, Ann Oncol 10 Suppl 4:60-3). In addition,angiogenesis inhibitors have been shown to be more effective whencombined with radiation and chemotherapeutic agents (Klement, 2000, J.Clin Invest, 105(8) R15-24. Browder, 2000, Cancer Res. 6-(7) 1878-86,Arap et al., 1998, Science 279(5349):377-80; Mauceri et al., 1998,Nature 394(6690):287-91).

Cancer is not only devastating to humans, but is also the most commoncause of natural death in dogs. (Bronson, 1982, Am J Vet Res, 43(11)2057-9). Dogs develop tumors twice as frequently as humans and it hasbeen reported that 45-50% of dogs that live to 10 years or older die ofcancer; regardless of age, and that 23% of dogs that present fornecropsy died of cancer(Bronson, 1982, Am J Vet Res, 43(11) 2057-9).Surgical removal of the tumor is the most common treatment, but theprognosis for invasive/metastatic tumor is very poor, with mediansurvival time ranging from weeks to months. Other treatments, such asradiation therapy and chemotherapy, have only very limited success(Bostock, 1986, Br Vet J 142(6):506-15; Bostock, 1986, Br Vet J142(1):1-19; MacEwen, 1990, Cancer Metastasis Rev 9(2): 125-36). Thus,more effective treatments for angiogenic diseases, such as, for example,canine cancers, are necessary.

SUMMARY OF THE INVENTION

The present invention encompasses novel nucleotide sequences that areassociated with angiogenesis related disorders, e.g., cancer. Theinvention more specifically relates to nucleotide sequences that encodeendostatin. In addition, endostatin nucleic acids, recombinant DNAmolecules, cloned genes or degenerate variants thereof are providedherein. The invention also provides vectors, including expressionvectors, containing endostatin nucleic acid molecules, and hosts thathave been genetically engineered to express and/or contain suchendostatin gene products.

The invention further relates to novel endostatin gene products and toantibodies directed against such gene products, or variants or fragmentsthereof.

The invention further relates to methods for modulation ofendostatin-mediated processes and for the treatment of disordersinvolving angiogenesis, such as cancer, including the amelioration orprevention of at least one symptom of the disorders, wherein suchmethods comprise administering a compound which modulates the expressionof an endostatin gene and/or the synthesis or activity of an endostatingene product. In one embodiment, the invention relates to methods forthe use of a novel endostatin gene product or fragment, analog, ormimetic thereof, or an antibody or antibody fragment directed against anendostatin gene product, to treat or ameliorate a symptom of suchdisorders.

Such disorders include, but are not limited to, angiogenesis-dependentcancer, including, for example, solid tumors, blood born tumors such asleukemias, and tumor metastases; benign tumors, for example hemangiomas,acoustic neuromas, neurofibromas, trachomas, and pyogenic granulomas;rheumatoid arthritis; psoriasis; ocular angiogenic diseases, forexample, diabetic retinopathy, retinopathy of prematurity, maculardegeneration, corneal graft rejection, neovascular glaucoma, retrolentalfibroplasia, rubeosis; Osler-Webber Syndrome; myocardial angiogenesis;plaque neovascularization; telangiectasia; hemophiliac joints;angiofibroma; wound granulation; corornary collaterals; cerebralcollaterals; arteriovenous malformations; ischemic limb angiogenesis;diabetic neovascularization; macular degeneration; fractures;vasculogenesis; hematopoiesis; ovulation; menstruation; andplacentation.

The invention further relates to methods for modulation ofendostatin-mediated processes and for the treatment of disordersinvolving abnormal stimulation of endothelial cells, including theamelioration or prevention of at least one symptom of the disorders,wherein such methods comprise administering a compound which modulatesthe expression of an endostatin gene and/or the synthesis or activity ofan endostatin gene product. In one embodiment, the invention relates tomethods for the use of a novel endostatin gene product or fragment,analog, or mimetic thereof, or an antibody or antibody fragment directedagainst an endostatin gene product, to treat or ameliorate a symptom ofsuch disorders.

The endothelial cell proliferation inhibiting proteins of the presentinvention are useful in the treatment of disease of excessive orabnormal stimulation of endothelial cells. These diseases include, butare not limited to, intestinal adhesions, atherosclerosis, scleroderma,and hypertrophic scars, i.e., keloids. They are also useful in thetreatment of diseases that have angiogenesis as a pathologic consequencesuch as cat scratch disease (Rochele minalia quintosa) and ulcers(Helobacter pylori).

The invention further relates to methods for blocking interactionsbetween endostatin and its respective receptors with analogs that act asreceptor antagonists. These antagonists may promote endothelializationand vascularization. Such effects may be desirable in situationsincluding, but not limited to, inadequate vascularization of the uterineendometrium and associated infertilty, wound repair, healing of cuts andincisions, treatment of vascular problems in diabetics, especiallyretinal and peripheral vessels, promotion of vascularization intransplanted tissue including muscle and skin, promotion ofvascularization of cardiac muscle especially following transplantationof a heart or heart tissue and after bypass surgery, promotion ofvascularization of solid and relatively avascular tumors for enhancedcytotoxin delivery, and enhancement of blood flow to the nervous system,including but not limited to the cerebral cortex and spinal cord.

The term “endostatin-related disorder” as used herein, refers todisorders involving an endostatin gene or gene product, or an aberrantlevel of endostatin gene expression, gene product synthesis and/or geneproduct activity, respectively, relative to levels found in normal,unaffected, unimpaired individuals, levels found in clinically normalindividuals, and/or levels found in a population whose levels representbaseline, average endostatin levels.

The term “endostatin-mediated process” as used herein, includesprocesses dependent and/or responsive, either directly or indirectly, tothe level of expression, gene product synthesis and/or gene productactivity of endostatin genes.

In another embodiment, such methods can comprise modulating the level ofexpression or the activity of an endostatin gene product in a cell suchthat the endostatin-mediated process or the disorder is treated, e.g., asymptom is ameliorated. In another embodiment, such methods can comprisesupplying a nucleic acid molecule encoding an endostatin gene product toincrease the level, expression or activity of the endostatin geneproduct within the cell such that the endostatin-mediated process or thedisorder is treated, e.g., a symptom is ameliorated. The nucleic acidmolecule encoding the endostatin gene product can encode a mutantendostatin gene product with increased activity or expression levels.

The invention still further relates to methods for modulation ofendostatin-mediated processes or the treatment of endostatin-relateddisorders, such as cancer, including, but not limited to, disordersresulting from endostatin gene mutations, and/or an abnormal levels ofendostatin expression or activity and disorders involving one or moreendostatin genes or gene products, wherein treatment includes theamelioration or prevention of at least one symptom of such disorders. Inone embodiment, such methods can comprise supplying a mammal in need oftreatment with a nucleic acid molecule encoding an unimpaired endostatingene product such that the unimpaired endostatin gene product isexpressed and the disorder is treated, e.g., a symptom is ameliorated.In another embodiment, such methods can comprise supplying a mammal inneed of treatment with a cell comprising a nucleic acid molecule thatencodes an unimpaired endostatin gene product such that the cellexpresses the unimpaired endostatin gene product and the disorder istreated, e.g., a symptom is ameliorated. In yet another embodiment, suchmethods comprise supplying a mammal in need of treatment with amodulatory compound, such as, for example, a small molecule, peptide orantibody that is capable of modulating the activity of an endostatingene or gene product.

In addition, the present invention is directed to methods that utilizeendostatin gene sequences and/or endostatin gene product sequences forthe diagnostic evaluation, genetic testing and/or prognosis ofangiogenesis-related disorders, such as cancer. For example, theinvention relates to methods for diagnosing angiogenesis-relateddisorders, e.g., cancer, wherein such methods can comprise measuringendostatin gene expression in a patient sample, or detecting anendostatin mutation that correlates with the presence or development ofsuch a disorder, in the genome of a mammal suspected of exhibiting sucha disorder.

The present invention also is directed to utilizing the endostatin genesequences and/or gene products as markers for mapping of the humanchromosome.

The invention still further relates to methods for identifying compoundscapable of modulating the expression of an endostatin gene and/or thesynthesis or activity of an endostatin gene product, wherein suchmethods comprise contacting a compound with a cell that expresses suchan endostatin gene, measuring the levels of endostatin gene expression,gene product expression or gene product activity, and comparing suchlevels to the levels of endostatin gene expression, gene product, orgene product activity produced by the cell in the absence of suchcompound, such that if the level obtained in the presence of thecompound differs from that obtained in its absence, a compound capableof modulating the expression of the endostatin gene and/or the synthesisor activity of the endostatin gene product has been identified.

Definitions

As used herein, the following terms shall have the abbreviationsindicated.

BAC: bacterial artificial chromosome

bp: base pair(s)

dbEST: expressed sequence tag data base (National Center forBiotechnology Information)

EST: expressed sequence tag

RT-PCR: reverse transcriptase PCR

SSCP: single-stranded conformational polymorphism

SNP: single nucleotide polymorphism

YAC: yeast artificial chromosome

BRIEF DESCRIPTION OF THE FIGURES

FIG. 1: RT-PCR analysis of dog liver RNA. FIG. 1 shows results of anamplification reaction of dog liver RNA. A region of canine collagenXVIII which contains endostatin (pro-endo) were specifically amplified.The positions of PCR products of expected size are indicated by anarrow.

FIG. 2: The nucleotide sequence of canine pro-endostatin (SEQ ID NO:1).

FIG. 3: The amino acid sequence of canine pro-endostatin translated fromthe sequence of FIG. 2 (SEQ ID NO:2). The region corresponding toendostatin is in bold (amino acid residues 47-230). The stop codon isindicated by *.

FIG. 4: The nucleotide sequence of canine endostatin (SEQ ID NO:3).

FIG. 5: The amino acid sequence of canine endostatin translated from thesequence of FIG. 4 (SEQ ID NO:4). The stop codon is indicated by *.

FIG. 6: An amino acid alignment of endostatin from canine, chicken,human and mouse. The program used is Lasergene MegAlign, aligned byClustal method (DNA Star Inc., Madision, Wis.).

FIG. 7: Immunofluorescence analysis of canine and murine endostatin. 293cells were transfected with HA-tagged canine endostatin (ca-endo) andmurine endostatin (mu-endo). The cells were stained with antibodyagainst the HA epitope and TRITC conjugated secondary antibody.

FIG. 8: Immunoblot analysis of 293 cells transfected with HA-taggedcanine endostatin (ca-endo) and murine endostatin (mu-endo).Intracellular proteins from cell lysates and secreted proteins fromculture supernatants (sup) were run on SDS-PAGE and analyzed byimmunoblot using HA antibody and alkaline phosphatase conjugatedsecondary antibody.

FIG. 9: Endothelial Cell Proliferation Assay. The figure shows a summaryof inhibition of endothelial cell proliferation by HA-tagged canineendostatin (HA-ca-endo) and murine endostatin (HA-mu-endo). 293 cellswere transfected with angiogenesis inhibitors or green fluorescentprotein (GFP) as control. The supernatants were harvested 48 hours posttransfection and incubated with bFGF stimulated CPAE bovine endothelialcells or 293 cells for 72 hours. The total numbers of cells were countedand plotted. Four independent experiments were carried out and eachexperiment was done in duplicate.

FIG. 10: Endothelial Cell Proliferation Assay. The figure shows asummary of inhibition of endothelial cell proliferation by untaggedcanine endostatin (ca-endo). 293 cells were transfected withangiogenesis inhibitors or green fluorescent protein (GFP) as control.The supernatants were harvested 48 hours post transfection and incubatedwith bFGF stimulated CPAE bovine endothelial cells or 293 cells for 72hours. The total numbers of cells were counted and plotted. Threeindependent experiments were carried out and each experiment was done induplicate.

DETAILED DESCRIPTION OF THE INVENTION

Compositions and methods relating to nucleic acid sequences associatedwith disorders involving angiogenesis are described herein. Novel geneswhich are associated with angiogenesis-related disorders have beenidentified. Such genes encode endostatin. In particular, described beloware endostatin nucleic acid molecules, as well as vectors comprisingthese molecules, host cells engineered to contain and/or express suchmolecules, endostatin gene products, and antibodies that specificallyrecognize such gene products. Also described are various uses of thesenucleic acids, polypeptides, and antibodies, as well as methods fortheir detection. For example, methods for the use of these molecules formodulation of angiogenesis-related processes and for treatment ofangiogenesis-related disorders, such as cancer, are described. Screeningassays for compounds that interact with an endostatin gene or geneproduct, or modulate endostatin gene or gene product activity also aredescribed below. Methods of treatment of an angiogenesis-relateddisorder using the compositions of the invention and compositionsidentified by the methods of the invention are further described.Finally, pharmaceutical compositions for use with the compositions ofthe invention are described.

Endostatin nucleic acid molecules are described in this section. Unlessotherwise stated, the term “endostatin nucleic acid” refers collectivelyto the sequences described herein.

The endostatin nucleic acid molecules of the invention include:

-   -   (a) a nucleic acid molecule containing the DNA sequence of        endostatin (FIG. 4 (SEQ ID NO:3)) and fragments thereof;    -   (b) a nucleic acid molecule comprising an endostatin nucleic        acid sequence (e.g., the nucleic acid sequences depicted in FIG.        2 (SEQ ID NO:1)) or a fragment thereof;    -   (c) a nucleic acid molecule that encodes an endostatin gene        product;    -   (d) a nucleic acid molecule that comprises at least one exon of        an endostatin gene;    -   (e) a nucleic acid molecule that comprises endostatin gene        sequences of upstream untranslated regions, intronic regions,        and/or downstream untranslated regions, or fragments thereof, of        the endostatin nucleotide sequences in (b) above;    -   (f) a nucleic acid molecule comprising the novel endostatin        sequences disclosed herein that encodes mutants of the        endostatin gene products in which all or a part of one or more        of the domains is deleted or altered, as well as fragments        thereof;    -   (g) nucleic acid molecules that encode fusion proteins        comprising an endostatin gene product, or a fragment thereof,        fused to a heterologous polypeptide;    -   (h) nucleic acid molecules within the endostatin genes described        in b), above (e.g., primers), or within chromosomal nucleotide        sequences flanking the endostatin gene, which can be utilized as        part of the methods of the invention for identifying and        diagnosing individuals at risk for, or exhibiting an        angiogenesis-related disorder, such as cancer, or can be used        for mapping human chromosomes; and;    -   (i) nucleic acid molecules within the endostatin genes described        in b), above, or within chromosomal nucleotide sequences        flanking the endostatin genes, which correlate with an        angiogenesis-related disorder, such as cancer.

The endostatin nucleotide sequences of the invention further includenucleotide sequences corresponding to the nucleotide sequences of(a)-(i) above wherein one or more of the exons, or fragments thereof,have been deleted.

The endostatin nucleotide sequences of the invention also includenucleotide sequences greater than 20, 30, 40, 50, 60, 70, 80, 90, 100,or more base pairs long that have at least 85%, 90%, 95%, 98%, or morenucleotide sequence identity to the endostatin nucleotide sequences of(a)-(i) above, with the proviso that the endostatin is not chicken,human, or mouse endostatin.

The endostatin nucleotide sequences of the invention further includenucleotide sequences that encode polypeptides having at least 85%, 90%,95%, 98%, or higher amino acid sequence identity to the polypeptidesencoded by the endostatin nucleotide sequences of (a)-(i) above.

To determine the percent identity of two amino acid sequences or of twonucleic acids, the sequences are aligned for optimal comparison purposes(e.g., gaps can be introduced in the sequence of a first amino acid ornucleic acid sequence for optimal alignment with a second amino ornucleic acid sequence). The amino acid residues or nucleotides atcorresponding amino acid positions or nucleotide positions are thencompared. When a position in the first sequence is occupied by the sameamino acid residue or nucleotide as the corresponding position in thesecond sequence, then the molecules are identical at that position. Thepercent identity between the two sequences is a function of the numberof identical positions shared by the sequences (i.e., % identity=# ofidentical overlapping positions/total # of overlapping positions×100%).In one embodiment, the two sequences are the same length.

The determination of percent identity between two sequences can also beaccomplished using a mathematical algorithm. A preferred, non-limitingexample of a mathematical algorithm utilized for the comparison of twosequences is the algorithm of Karlin and Altschul, 1990, Proc. Natl.Acad. Sci. USA 87:2264-2268, modified as in Karlin and Altschul, 1993,Proc. Natl. Acad. Sci. USA 90:5873-5877. Such an algorithm isincorporated into the NBLAST and XBLAST programs of Altschul, et al.,1990, J. Mol. Biol. 215:403-410. BLAST nucleotide searches can beperformed with the NBLAST program, score=100, wordlength=12 to obtainnucleotide sequences homologous to a nucleic acid molecules of theinvention. BLAST protein searches can be performed with the XBLASTprogram, score=50, wordlength=3 to obtain amino acid sequenceshomologous to a protein molecules of the invention. To obtain gappedalignments for comparison purposes, Gapped BLAST can be utilized asdescribed in Altschul et al., 1997, Nucleic Acids Res.25:3389-3402.Alternatively, PSI-Blast can be used to perform an iterated search whichdetects distant relationships between molecules (Altschul et al., 1997,supra). When utilizing BLAST, Gapped BLAST, and PSI-Blast programs, thedefault parameters of the respective programs (e.g., XBLAST and NBLAST)can be used (see http://www.ncbi.nlm.nih.gov). Another preferred,non-limiting example of a mathematical algorithm utilized for thecomparison of two sequences is the algorithm of Karlin and Altschul,1990, Proc. Natl. Acad. Sci. USA 87:2264-2268, modified as in Karlin andAltschul, 1993, Proc. Natl. Acad. Sci. USA 90:5873-5877. Such analgorithm is incorporated into the NBLAST and XBLAST programs ofAltschul, et al., 1990, J. Mol. Biol. 215:403-410. BLAST nucleotidesearches can be performed with the NBLAST program, score=100,wordlength=12 to obtain nucleotide sequences homologous to a nucleicacid molecules of the invention. BLAST protein searches can be performedwith the XBLAST program, score=50, wordlength=3 to obtain amino acidsequences homologous to a protein molecules of the invention. To obtaingapped alignments for comparison purposes, Gapped BLAST can be utilizedas described in Altschul et al., 1997, Nucleic Acids Res.25:3389-3402.Alternatively, PSI-Blast can be used to perform an iterated search whichdetects distant relationships between molecules (Altschul et al., 1997,supra). When utilizing BLAST, Gapped BLAST, and PSI-Blast programs, thedefault parameters of the respective programs (e.g., XBLAST and NBLAST)can be used (see http://www.ncbi.nlm.nih.gov). Another preferred,non-limiting example of a mathematical algorithm utilized for thecomparison of sequences is the algorithm of Myers and Miller, 1988,CABIOS 4:11-17. Such an algorithm is incorporated into the ALIGN program(version 2.0) which is part of the GCG sequence alignment softwarepackage. When utilizing the ALIGN program for comparing amino acidsequences, a PAM120 weight residue table, a gap length penalty of 12,and a gap penalty of 4 can be used.

The percent identity between two sequences can be determined usingtechniques similar to those described above, with or without allowinggaps. In calculating percent identity, typically only exact matches arecounted.

The endostatin nucleotide sequences of the invention further include:(a) any nucleotide sequence that hybridizes to an endostatin nucleicacid molecule of the invention under stringent conditions, e.g.,hybridization to filter-bound DNA in 6× sodium chloride/sodium citrate(SSC) at about 45° C. followed by one or more washes in 0.2×SSC/0.1% SDSat about 50-65° C., or (b) under highly stringent conditions, e.g.,hybridization to filter-bound nucleic acid in 6×SSC at about 45° C.followed by one or more washes in 0.1×SSC/0.2% SDS at about 68° C., orunder other hybridization conditions which are apparent to those ofskill in the art (see, for example, Ausubel F. M. et al., eds., 1989,Current Protocols in Molecular Biology, Vol. I, Green PublishingAssociates, Inc., and John Wiley & sons, Inc., New York, at pp.6.3.1-6.3.6 and 2.10.3). Preferably the endostatin nucleic acid moleculethat hybridizes under conditions described under (a) and (b), above, isone that comprises the complement of a nucleic acid molecule thatencodes an endostatin gene product. In a preferred embodiment, nucleicacid molecules that hybridize under conditions (a) and (b), above,encode gene products, e.g., gene products functionally equivalent to anendostatin gene product.

Functionally equivalent endostatin gene products include naturallyoccurring endostatin gene products present in the same or differentspecies. Functionally equivalent endostatin gene products also includegene products that retain at least one of the biological activities ofan endostatin gene product, and/or which are recognized by and bind toantibodies (polyclonal or monoclonal) directed against such geneproduct.

Among the nucleic acid molecules of the invention aredeoxyoligonucleotides (“oligos”) which hybridize under highly stringentor stringent conditions to the endostatin nucleic acid moleculesdescribed above. In general, for probes between 14 and 70 nucleotides inlength the melting temperature (Tm) is calculated using the formula:Tm(° C.)=81.5+16.6 (log [monovalent cations (molar)])+0.41 (%G+C)−(500/N) where N is the length of the probe. If the hybridization iscarried out in a solution containing formamide, the melting temperatureis calculated using the equation Tm(° C.)=81.5+16.6 (log[monovalentcations (molar)])+0.41(% G+C)−(0.61% formamide)−(500/N) where N is thelength of the probe. In general, hybridization is carried out at about20-25 degrees below Tm (for DNA-DNA hybrids) or 10-15 degrees below Tm(for RNA-DNA hybrids).

Exemplary highly stringent conditions may refer, e.g., to washing in6×SSC/0.05% sodium pyrophosphate at 37° C. (for about 14-base oligos),48° C. (for about 17-base oligos), 55° C. (for about 20-base oligos),and 60° C. (for about 23-base oligos).

The nucleic acid molecules of the invention further comprise thecomplements of the nucleic acids described above. Such molecules can,for example, act as antisense molecules, useful, for example, inendostatin gene regulation, and/or as antisense primers in amplificationreactions of endostatin gene nucleic acid sequences.

The nucleic acid sequences of the invention may be used as part ofribozyme and/or triple helix sequences, also useful for endostatin generegulation. Still further, such molecules may be used as components ofdiagnostic methods whereby, for example, the presence of a particularendostatin allele involved in an angiogenesis-related disorder, e.g.,cancer, may be detected, or whereby the methods involve mapping thehuman chromosomal region spanned by the alleles.

Fragments of the endostatin nucleic acid molecules refer to endostatinnucleic acid sequences that can be at least 10, 12, 15, 20, 30, 40, 50,60, 70, 80, 90, 100, 200, 300, 400, 500, 600, 700, 800, 900, 1000, 1250,1500, 1750, 2000, 2250, 2500, 2750, 3000, 3250, 3500, 3750, 4000, 4250,4500, 4750, 5000, or more contiguous nucleotides in length.Alternatively, the fragments can comprise sequences that encode at least10, 20, 30, 40, 50, 100, 150, 200, 250, 300, 350, 400, 450 or morecontiguous amino acid residues of the endostatin gene products. In oneembodiment, the endostatin nucleic acid molecules encode a gene productexhibiting at least one biological activity of a correspondingendostatin gene product, e.g., an endostatin gene product. Fragments ofthe endostatin nucleic acid molecules can refer also to endostatin exonsor introns, and, further, can refer to portions of endostatin codingregions that encode domains of endostatin gene products.

With respect to identification and isolation of endostatin nucleotidesequences, such sequences can be readily obtained, for example, byutilizing standard sequencing and bacterial artificial chromosome (BAC)technologies.

As will be appreciated by those skilled in the art, DNA sequencepolymorphisms of an endostatin gene will exist within a population ofindividual organisms (e.g., within a human or canine population). Suchpolymorphisms may exist, for example, among individual organisms withina population due to natural allelic variation. Such polymorphismsinclude ones that lead to changes in amino acid sequence. As usedherein, the phrase “allelic variant” refers to a nucleotide sequencewhich occurs at a given locus or to a gene product encoded by thatnucleotide sequence. Such natural allelic variations can result in 1-5%,5-20%, or 20-50% variance in the nucleotide sequence of a given gene. Anallele is one of a group of genes which occur alternatively at a givengenetic locus. Alternative alleles can be identified by sequencing thegene of interest in a number of different individual organisms. This canbe readily carried out by using hybridization probes to identify thesame genetic locus in a variety of individual organisms. As used herein,the terms “gene” and “recombinant gene” refer to nucleic acid moleculescomprising an open reading frame encoding a polypeptide of theinvention. The term can further include nucleic acid moleculescomprising upstream and/or exon/intron sequences and structure.

With respect to the cloning of additional allelic variants of the humanendostatin gene and homologs and orthologs from other species (e.g.,guinea pig, cow, mouse, canine), the isolated endostatin gene sequencesdisclosed herein may be labeled and used to screen a cDNA libraryconstructed from mRNA obtained from appropriate cells or tissues (e.g.,liver) derived from the organism (e.g., guinea pig, cow, mouse, canine)of interest. The hybridization conditions used should generally be of alower stringency when the cDNA library is derived from an organismdifferent from the type of organism from which the labeled sequence wasderived, and can routinely be determined based on, e.g., relativerelatedness of the target and reference organisms.

Alternatively, the labeled fragment may be used to screen a genomiclibrary derived from the organism of interest, again, usingappropriately stringent conditions. Appropriate stringency conditionsare well known to those of skill in the art as discussed above, and willvary predictably depending on the specific organisms from which thelibrary and the labeled sequences are derived. For guidance regardingsuch conditions see, for example, Sambrook, et al., 1989, MolecularCloning, A Laboratory Manual, Second Edition, Cold Spring Harbor Press,N.Y.; and Ausubel, et al., 1989-1999, Current Protocols in MolecularBiology, Green Publishing Associates and Wiley Interscience, N.Y., bothof which are incorporated herein by reference in their entirety.

Further, an endostatin gene allelic variant may be isolated from, forexample, human or canine nucleic acid, by performing PCR using twodegenerate oligonucleotide primer pools designed on the basis of aminoacid sequences within the endostatin gene products disclosed herein. Thetemplate for the reaction may be cDNA obtained by reverse transcriptionof mRNA prepared from, for example, human or non-human cell lines ortissue known or suspected to express a wild type or mutant endostatingene allele (such as, for example, liver cells). In one embodiment, theallelic variant is isolated from an individual organism that has anangiogenesis-mediated disorder.

The PCR product may be subcloned and sequenced to ensure that theamplified sequences represent the sequences of an endostatin genenucleic acid sequence. The PCR fragment may then be used to isolate afull length cDNA clone by a variety of methods. For example, theamplified fragment may be labeled and used to screen a bacteriophagecDNA library. Alternatively, the labeled fragment may be used to isolategenomic clones via the screening of a genomic library.

PCR technology also may be utilized to isolate full length cDNAsequences, as well as cDNA sequences corresponding to alternativelyspliced mRNA species. For example, RNA may be isolated, followingstandard procedures, from an appropriate cellular or tissue source(i.e., one known, or suspected, to express the endostatin gene, such as,for example, liver tissue samples obtained through biopsy orpost-mortem). A reverse transcription reaction may be performed on theRNA using an oligonucleotide primer specific for the most 5′ end of theamplified fragment for the priming of first strand synthesis. Theresulting RNA/DNA hybrid may then be “tailed” with guanines using astandard terminal transferase reaction, the hybrid may be digested withRNase H, and second strand synthesis may then be primed with a poly-Cprimer. Thus, cDNA sequences upstream of the amplified fragment mayeasily be isolated. For a review of cloning strategies that may be used,see e.g., Sambrook et al., 1989, supra, or Ausubel et al., supra.

A cDNA of an allelic, e.g., mutant, variant of the endostatin gene maybe isolated, for example, by using PCR, a technique that is well knownto those of skill in the art. In this case, the first cDNA strand may besynthesized by hybridizing an oligo-dT oligonucleotide to mRNA isolatedfrom tissue known or suspected to be expressed in an individual organismputatively carrying a mutant endostatin allele, and by extending the newstrand with reverse transcriptase. The second strand of the cDNA is thensynthesized using an oligonucleotide that hybridizes specifically to the5′ end of the normal gene. Using these two primers, the product is thenamplified via PCR, cloned into a suitable vector, and subjected to DNAsequence analysis through methods well known to those of skill in theart. By comparing the DNA sequence of the mutant endostatin allele tothat of the normal endostatin allele, the mutation(s) responsible forthe loss or alteration of function of the mutant endostatin gene productcan be ascertained.

Alternatively, a genomic library can be constructed using DNA obtainedfrom an individual organism suspected of or known to carry a mutantendostatin allele, or a cDNA library can be constructed using RNA from atissue known, or suspected, to express a mutant endostatin allele. Anunimpaired endostatin gene, or any suitable fragment thereof, may thenbe labeled and used as a probe to identify the corresponding mutantendostatin allele in such libraries. Clones containing the mutantendostatin gene sequences may then be purified and subjected to sequenceanalysis according to methods well known to those of skill in the art.

Additionally, an expression library can be constructed utilizing cDNAsynthesized from, for example, RNA isolated from a tissue known, orsuspected, to express a mutant endostatin allele in an individualorganism suspected of or known to carry such a mutant allele. In thismanner, gene products made by the putatively mutant tissue may beexpressed and screened using standard antibody screening techniques inconjunction with antibodies raised against the normal endostatin geneproduct, as described, below. (For screening techniques, see, forexample, Harlow and Lane, eds., 1988, “Antibodies: A Laboratory Manual”,Cold Spring Harbor Press, Cold Spring Harbor.)

In cases where an endostatin mutation results in an expressed geneproduct with altered function (e.g., as a result of a missense or aframeshift mutation), a polyclonal set of anti-endostatin gene productantibodies are likely to cross-react with the mutant endostatin geneproduct. Library clones detected via their reaction with such labeledantibodies can be purified and subjected to sequence analysis accordingto methods well known to those of skill in the art.

Endostatin mutations or polymorphisms can further be detected using PCRamplification techniques. Primers can routinely be designed to amplifyoverlapping regions of the whole endostatin sequence including thepromoter regulating region. In one embodiment, primers are designed tocover the exon-intron boundaries such that, coding regions can bescanned for mutations.

The invention also includes nucleic acid molecules, preferably DNAmolecules, that are the complements of the nucleotide sequences of thepreceding paragraphs.

In certain embodiments, the nucleic acid molecules of the invention arepresent as part of nucleic acid molecules comprising nucleic acidsequences that contain or encode heterologous (e.g., vector, expressionvector, or fusion protein) sequences.

Endostatin gene products include those gene products encoded by nucleicacid molecules comprising the endostatin gene sequences described,above. In addition, endostatin gene products may include proteins thatrepresent functionally equivalent gene products. Such an equivalentendostatin gene products may contain deletions, including internaldeletions, additions, including additions yielding fusion proteins, orsubstitutions of amino acid residues within and/or adjacent to the aminoacid sequence encoded by the endostatin gene sequences described, above,but that result in a “silent” change, in that the change produces afunctionally equivalent endostatin gene product. Amino acidsubstitutions may be made on the basis of similarity in polarity,charge, solubility, hydrophobicity, hydrophilicity, and/or theamphipathic nature of the residues involved. For example, nonpolar(hydrophobic) amino acids include alanine, leucine, isoleucine, valine,proline, phenylalanine, tryptophan, and methionine; polar neutral aminoacids include glycine, serine, threonine, cysteine, tyrosine,asparagine, and glutamine; positively charged (basic) amino acidsinclude arginine, lysine, and histidine; and negatively charged (acidic)amino acids include aspartic acid and glutamic acid.

Alternatively, where alteration of function is desired, deletion ornon-conservative alterations can be engineered to produce alteredendostatin gene products. Such alterations can, for example, alter oneor more of the biological functions of the endostatin gene product.Further, such alterations can be selected so as to generate endostatingene products that are better suited for expression, scale up, etc. inthe host cells chosen. For example, cysteine-residues can be deleted orsubstituted with another amino acid residue in order to eliminatedisulfide bridges.

Peptides and/or proteins corresponding to one or more domains of anendostatin protein, as well as fusion proteins, in which an endostatinprotein or a portion of an endostatin protein, such as a truncatedendostatin protein, or peptide or an endostatin protein domain, is fusedto an unrelated protein are also within the scope of this invention.Such proteins and peptides can be designed on the basis of theendostatin nucleotide sequence disclosed, above, and/or on the basis ofthe endostatin amino acid sequence disclosed herein. Fusion proteinsinclude, but are not limited to, IgFc fusions which stabilize theendostatin protein or peptide and prolong half life in vivo; or fusionsto any amino acid sequence that allows the fusion protein to be anchoredto the cell membrane; or fusions of endostatin protein domains to anenzyme, fluorescent protein, luminescent protein, or a flag epitopeprotein or peptide which provides a marker function.

Endostatin proteins of the invention also include endostatin proteinsequences wherein domains encoded by at least one exon of the cDNAsequence, or fragments thereof, have been deleted.

The endostatin protein sequences described above can include a domainwhich comprises a signal sequence that targets the endostatin geneproduct for secretion. As used herein, a signal sequence includes apeptide of at least about 15 or 20 amino acid residues in length whichoccurs at the N-terminus of secretory and membrane-bound proteins andwhich contains at least about 70% hydrophobic amino acid residues suchas alanine, leucine, isoleucine, phenylalanine, proline, tyrosine,tryptophan, or valine. In a preferred embodiment, a signal sequencecontains at least about 10 to 40 amino acid residues, preferably about19-34 amino acid residues, and has at least about 60-80%, morepreferably 65-75%, and more preferably at least about 70% hydrophobicresidues. A signal sequence serves to direct a protein containing such asequence to a lipid bilayer.

A signal sequence of a polypeptide of the invention can be used tofacilitate secretion and isolation of the secreted protein or otherproteins of interest. Signal sequences are typically characterized by acore of hydrophobic amino acids which are generally cleaved from themature protein during secretion in one or more cleavage events. Suchsignal peptides contain processing sites that allow cleavage of thesignal sequence from the mature proteins as they pass through thesecretory pathway. Thus, the invention pertains to the describedendostatin polypeptides having a signal sequence (that is, “immature”polypeptides), as well as to the endostatin signal sequences themselvesand to the endostatin polypeptides in the absence of a signal sequence(i.e., the “mature” endostatin cleavage products). It is to beunderstood that endostatin polypeptides of the invention can furthercomprise polypeptides comprising any signal sequence havingcharacteristics as described above and a mature endostatin polypeptidesequence.

The endostatin polypeptides of the invention can further compriseposttranslational modifications, including, but not limited to,glycosylations, acetylations, myristylations, and phosphorylations. Ifthe native endostatin protein does not have recognition motifs thatallow such modifications, it would be routine for one skilled in the artto introduce into an endostatin gene nucleotide sequences that encodemotifs such as enzyme recognition signals so as to produce a modifiedendostatin gene product.

The endostatin gene products, peptide fragments thereof and fusionproteins thereof, may be produced by recombinant DNA technology usingtechniques well known in the art. Thus, methods for preparing theendostatin gene polypeptides, peptides, fusion peptide and fusionpolypeptides of the invention by expressing nucleic acid containingendostatin gene sequences are described herein. Methods that are wellknown to those skilled in the art can be used to construct expressionvectors containing endostatin gene product coding sequences andappropriate transcriptional and translational control signals. Thesemethods include, for example, in vitro recombinant DNA techniques,synthetic techniques, and in vivo genetic recombination. See, e.g., thetechniques described in Sambrook, et al., 1989, supra, and Ausubel, etal., 1989, supra. Alternatively, RNA capable of encoding endostatin geneproduct sequences may be chemically synthesized using, for example,synthesizers. See, e.g., the techniques described in “OligonucleotideSynthesis”, 1984, Gait, ed., IRL Press, Oxford.

A variety of host-expression vector systems may be utilized to expressthe endostatin gene coding sequences of the invention. Suchhost-expression systems represent vehicles by which the coding sequencesof interest may be produced and subsequently purified, but alsorepresent cells that may, when transformed or transfected with theappropriate nucleotide coding sequences, exhibit the endostatin geneproduct of the invention in situ. These include, but are not limited to,microorganisms such as bacteria (e.g., E. coli, B. subtilis) transformedwith recombinant bacteriophage DNA, plasmid DNA or cosmid DNA expressionvectors containing endostatin gene product coding sequences; yeast(e.g., Saccharomyces, Pichia) transformed with recombinant yeastexpression vectors containing the endostatin gene product codingsequences; insect cell systems infected with recombinant virusexpression vectors (e.g., baculovirus) containing the endostatin geneproduct coding sequences; plant cell systems infected with recombinantvirus expression vectors (e.g., cauliflower mosaic virus, CaMV; tobaccomosaic virus, TMV) or transformed with recombinant plasmid expressionvectors (e.g., Ti plasmid) containing endostatin gene product codingsequences; or mammalian cell systems (e.g., COS, CHO, BHK, 293, 3T3)harboring recombinant expression constructs containing promoters derivedfrom the genome of mammalian cells (e.g., metallothionein promoter) orfrom mammalian viruses (e.g., the adenovirus late promoter; the vacciniavirus 7.5K promoter).

In bacterial systems, a number of expression vectors may beadvantageously selected depending upon the use intended for theendostatin gene product being expressed. For example, when a largequantity of such a protein is to be produced, for the generation ofpharmaceutical compositions of endostatin protein or for raisingantibodies to endostatin protein, for example, vectors that direct theexpression of high levels of fusion protein products that are readilypurified may be desirable. Such vectors include, but are not limited, tothe E. coli expression vector pUR278 (Ruther et al., 1983, EMBO J. 2,1791), in which the endostatin gene product coding sequence may beligated individually into the vector in frame with the lac Z codingregion so that a fusion protein is produced; pIN vectors (Inouye andInouye, 1985, Nucleic Acids Res. 13, 3101-3109; Van Heeke and Schuster,1989, J. Biol. Chem. 264, 5503-5509); and the like. pGEX vectors mayalso be used to express foreign polypeptides as fusion proteins withglutathione S-transferase (GST). In general, such fusion proteins aresoluble and can easily be purified from lysed cells by adsorption toglutathione-agarose beads followed by elution in the presence of freeglutathione. The pGEX vectors are designed to include thrombin or factorXa protease cleavage sites so that the cloned target gene product can bereleased from the GST moiety.

In an insect system, Autographa californica, nuclear polyhedrosis virus(AcNPV) is used as a vector to express foreign genes. The virus grows inSpodoptera frugiperda cells. Endostatin gene coding sequences may becloned individually into non-essential regions (for example thepolyhedrin gene) of the virus and placed under control of an AcNPVpromoter (for example the polyhedrin promoter). Successful insertion ofendostatin gene coding sequences will result in inactivation of thepolyhedrin gene and production of non-occluded recombinant virus (i.e.,virus lacking the proteinaceous coat coded for by the polyhedrin gene).These recombinant viruses are then used to infect Spodoptera frugiperdacells in which the inserted gene is expressed (e.g., see Smith, et al.,1983, J. Virol. 46, 584; Smith, U.S. Pat. No. 4,215,051).

In mammalian host cells, a number of viral-based expression systems maybe utilized. In cases where an adenovirus is used as an expressionvector, an endostatin gene coding sequence of interest may be ligated toan adenovirus transcription/translation control complex, e.g., the latepromoter and-tripartite leader sequence. This chimeric gene may then beinserted in the adenovirus genome by in vitro or in vivo recombination.Insertion in a non-essential region of the viral genome (e.g., region E1or E3) will result in a recombinant virus that is viable and capable ofexpressing endostatin gene product in infected hosts. (e.g., See Loganand Shenk, 1984, Proc. Natl. Acad. Sci. USA 81, 3655-3659). Specificinitiation signals may also be required for efficient translation ofinserted endostatin gene product coding sequences. These signals includethe ATG initiation codon and adjacent sequences. In cases where anentire endostatin gene, including an initiation codon and adjacentsequences, is inserted into the appropriate expression vector, noadditional translational control signals may be needed. In cases whereonly a portion of the endostatin gene coding sequence is inserted,similar exogenous translational control signals, including, perhaps, theATG initiation codon, must be provided. Furthermore, the initiationcodon must be in phase with the reading frame of the desired codingsequence to ensure translation of the entire insert. These exogenoustranslational control signals and initiation codons can be of a varietyof origins, both natural and synthetic. The efficiency of expression maybe enhanced by the inclusion of appropriate transcription enhancerelements, transcription terminators, etc. (see Bittner, et al., 1987,Methods in Enzymol. 153, 516-544).

In addition, a host cell strain may be chosen that modulates theexpression of the inserted sequences, or modifies and processes the geneproduct in the specific fashion desired. Such modifications (e.g.,glycosylation) and processing (e.g., cleavage) of protein products maybe important for the function of the protein. Different host cells havecharacteristic and specific mechanisms for the post-translationalprocessing and modification of proteins and gene products. Appropriatecell lines or host systems can be chosen to ensure the correctmodification and processing of the foreign protein expressed. To thisend, eukaryotic host cells that possess the cellular machinery forproper processing of the primary transcript, glycosylation, andphosphorylation of the gene product may be used. Such mammalian hostcells include but are not limited to CHO, VERO, BHK, HeLa, COS, MDCK,293, 3T3, and W138.

For long-term, high-yield production of recombinant proteins, stableexpression is preferred. For example, cell lines that stably express theendostatin gene product may be engineered. Rather than using expressionvectors that contain viral origins of replication, host cells can betransformed with DNA controlled by appropriate expression controlelements (e.g., promoter, enhancer, sequences, transcriptionterminators, polyadenylation sites, etc.), and a selectable marker.Following the introduction of the foreign DNA, engineered cells may beallowed to grow for 1-2 days in an enriched media, and then are switchedto a selective media. The selectable marker in the recombinant plasmidconfers resistance to the selection and allows cells to stably integratethe plasmid into their chromosomes and grow to form foci that in turncan be cloned and expanded into cell lines. This method mayadvantageously be used to engineer cell lines that express theendostatin gene product. Such engineered cell lines may be particularlyuseful in screening and evaluation of compounds that affect theendogenous activity of the endostatin gene product.

A number of selection systems may be used, including, but not limitedto, the herpes simplex virus thymidine kinase (Wigler, et al., 1977,Cell 11, 223), hypoxanthine-guanine phosphoribosyltransferase (Szybalskaand Szybalski, 1962, Proc. Natl. Acad. Sci. USA 48, 2026), and adeninephosphoribosyltransferase (Lowy, et al., 1980, Cell 22, 817) genes canbe employed in tk-, hgprt- or aprt-cells, respectively. Also,antimetabolite resistance can be used as the basis of selection for thefollowing genes: dhfr, which confers resistance to methotrexate (Wigler,et al., 1980, Natl. Acad. Sci. USA 77, 3567; O'Hare, et al., 1981, Proc.Natl. Acad. Sci. USA 78, 1527); gpt, which confers resistance tomycophenolic acid (Mulligan and Berg, 1981, Proc. Natl. Acad. Sci. USA78, 2072); neo, which confers resistance to the aminoglycoside G-418(Colberre-Garapin, et al., 1981, J. Mol. Biol. 150, 1); and hygro, whichconfers resistance to hygromycin (Santerre, et al., 1984, Gene 30, 147).

Alternatively, any fusion protein may be readily purified by utilizingan antibody specific for the fusion protein being expressed. Forexample, a system described by Janknecht, et al. allows for the readypurification of non-denatured fusion proteins expressed in human celllines (Janknecht, et al., 1991, Proc. Natl. Acad. Sci. USA 88,8972-8976). In this system, the gene of interest is subcloned into avaccinia recombination plasmid such that the gene's open reading frameis translationally fused to an amino-terminal tag consisting of sixhistidine residues. Extracts from cells infected with recombinantvaccinia virus are loaded onto Ni²⁺ nitriloacetic acid-agarose columnsand histidine-tagged proteins are selectively eluted withimidazole-containing buffers.

Alternatively, the expression characteristics of an endogenousendostatin gene within a cell, cell line, or microorganism may bemodified by inserting a heterologous DNA regulatory element into thegenome of a stable cell line or cloned microorganism such that theinserted regulatory element is operatively linked with the endogenousendostatin gene. For example, an endogenous endostatin gene which isnormally “transcriptionally silent”, i.e., an endostatin gene which isnormally not expressed, or is expressed only at very low levels in acell, cell line, or microorganism, may be activated by inserting aregulatory element which is capable of promoting the expression of anormally expressed gene product in that cell, cell line, ormicroorganism. Alternatively, a transcriptionally silent, endogenousendostatin gene may be activated by insertion of a promiscuousregulatory element that works across cell types.

A heterologous regulatory element may be inserted into a stable cellline or cloned microorganism, such that it is operatively linked with anendogenous endostatin gene, using techniques, such as targetedhomologous recombination, which are well known to those of skill in theart, and described e.g., in Chappel, U.S. Pat. No. 5,272,071; PCTpublication No. WO 91/06667, published May 16, 1991.

Endostatin gene products also can be expressed in transgenic animals.Animals of any species, including, but not limited to, mice, rats,rabbits, guinea pigs, pigs, micro-pigs, goats, sheep, and non-humanprimates, e.g., baboons, monkeys, and chimpanzees may be used togenerate endostatin transgenic animals. The term “transgenic,” as usedherein, refers to animals expressing endostatin gene sequences from adifferent species (e.g., mice expressing human or canine endostatinsequences), as well as animals that have been genetically engineered tooverexpress endogenous (i.e., same species) endostatin sequences oranimals that have been genetically engineered to no longer expressendogenous endostatin gene sequences (i.e., “knock-out” animals), andtheir progeny.

Any technique known in the art may be used to introduce an endostatingene transgene into animals to produce the founder lines of transgenicanimals. Such techniques include, but are not limited to, pronuclearmicroinjection (Hoppe and Wagner, 1989, U.S. Pat. No. 4,873,191);retrovirus mediated gene transfer into germ lines (van der Putten, etal., 1985, Proc. Natl. Acad. Sci., USA 82, 6148-6152); gene targeting inembryonic stem cells (Thompson, et al., 1989, Cell 56, 313-321);electroporation of embryos (Lo, 1983, Mol. Cell. Biol. 3, 1803-1814);and sperm-mediated gene transfer (Lavitrano et al., 1989, Cell 57,717-723). (For a review of such techniques, see Gordon, 1989, TransgenicAnimals, Intl. Rev. Cytol. 115, 171-229.)

Any technique known in the art may be used to produce transgenic animalclones containing an endostatin transgene, for example, nuclear transferinto enucleated oocytes of nuclei from cultured embryonic, fetal oradult cells induced to quiescence (Campbell, et al., 1996, Nature 380,64-66; Wilmut, et al., Nature 385, 810-813).

The present invention provides for transgenic animals that carry anendostatin transgene in all their cells, as well as animals that carrythe transgene in some, but not all their cells, i.e., mosaic animals.The transgene may be integrated as a single transgene or in concatamers,e.g., head-to-head tandems or head-to-tail tandems. The transgene mayalso be selectively introduced into and activated in a particular celltype by following, for example, the teaching of Lasko et al. (Lasko, etal., 1992, Proc. Natl. Acad. Sci. USA 89, 6232-6236). The regulatorysequences required for such a cell-type specific activation will dependupon the particular cell type of interest, and will be apparent to thoseof skill in the art. When it is desired that the endostatin genetransgene be integrated into the chromosomal site of the endogenousendostatin gene, gene targeting is preferred. Briefly, when such atechnique is to be utilized, vectors containing some nucleotidesequences homologous to the endogenous endostatin gene are designed forthe purpose of integrating, via homologous recombination withchromosomal sequences, into and disrupting the function of thenucleotide sequence of the endogenous endostatin gene. The transgene mayalso be selectively introduced into a particular cell type, thusinactivating the endogenous endostatin gene in only that cell type, byfollowing, for example, the teaching of Gu, et al. (Gu, et al., 1994,Science 265, 103-106). The regulatory sequences required for such acell-type specific inactivation will depend upon the particular celltype of interest, and will be apparent to those of skill in the art.

Once transgenic animals have been generated, the phenotypic expressionof the recombinant endostatin gene may be assayed utilizing standardtechniques. Initial screening may be accomplished by Southern blotanalysis or PCR techniques to analyze animal tissues to assay whetherintegration of the transgene has taken place. The level of mRNAexpression of the transgene in the tissues of the transgenic animals mayalso be assessed using techniques that include, but are not limited to,Northern blot analysis of tissue samples obtained from the animal, insitu hybridization analysis, and RT-PCR (reverse transcriptase PCR).Samples of endostatin gene-expressing tissue, may also be evaluatedimmunocytochemically using antibodies specific for the endostatintransgene product.

Endostatin gene products, or peptide fragments thereof, can be preparedfor a variety of uses. For example, such gene products, or peptidefragments thereof, can be used for the generation of antibodies, indiagnostic assays, or for mapping and the identification of othercellular or extracellular gene products involved in the regulation of anangiogenesis-related disorder, such as cancer. Such endostatin geneproducts include, but are not limited to, soluble derivatives such aspeptides or polypeptides corresponding to one or more domains of theendostatin gene product, particularly endostatin gene products that aremodified such that they are deleted for one or more hydrophobic domains.Alternatively, antibodies to the endostatin protein or anti-idiotypicantibodies that mimic the endostatin gene product (including Fabfragments), antagonists or agonists can be used to treatangiogenesis-related disorders, such as cancer. In yet another approach,nucleotide constructs encoding such endostatin gene products can be usedto genetically engineer host cells to express such endostatin geneproducts in vivo; these genetically engineered cells can function as“bioreactors” in the body delivering a continuous supply of endostatingene product, endostatin peptides or soluble endostatin polypeptides.

Described herein are methods for the production of antibodies capable ofspecifically recognizing one or more endostatin gene product epitopes orepitopes of conserved variants or peptide fragments of the geneproducts.

Such antibodies may include, but are not limited to, polyclonalantibodies, monoclonal antibodies (mAbs), canine, human, humanized orchimeric antibodies, single chain antibodies, Fab fragments, F(ab′)₂fragments, fragments produced by a Fab expression library,anti-idiotypic (anti-Id) antibodies, and epitope-binding fragments ofany of the above. Such antibodies may be used, for example, in thedetection of an endostatin gene product in an biological sample and may,therefore, be utilized as part of a diagnostic or prognostic techniquewhereby subjects may be tested for abnormal levels of endostatin geneproducts, and/or for the presence of abnormal forms of such geneproducts. Such antibodies may also be utilized in conjunction with, forexample, compound screening schemes, as described below, for theevaluation of the effect of test compounds on endostatin gene productlevels and/or activity. Additionally, such antibodies can be used inconjunction with the gene therapy techniques described below, forexample, to evaluate the normal and/or engineered endostatin-expressingcells prior to their introduction into the subject.

Anti-endostatin gene product antibodies may additionally be used as amethod for the inhibition of abnormal endostatin gene product activity.Thus, such antibodies may, be utilized as part of treatment methods foran angiogenesis-related disorder, e.g., cancer.

For the production of antibodies against an endostatin gene product,various host animals may be immunized by injection with an endostatingene product, or a portion thereof. Such host animals may include, butare not limited to, rabbits, mice, and rats, to name but a few. Variousadjuvants may be used to increase the immunological response, dependingon the host species, including but not limited to Freund's (complete andincomplete), mineral gels such as aluminum hydroxide, surface activesubstances such as lysolecithin, pluronic polyols, polyanions, peptides,oil emulsions, keyhole limpet hemocyanin, dinitrophenol, and potentiallyuseful human adjuvants such as BCG (bacille Calmette-Guerin) andCorynebacterium parvum.

Polyclonal antibodies are heterogeneous populations of antibodymolecules derived from the sera of animals immunized with an antigen,such as an endostatin gene product, or an antigenic functionalderivative thereof. For the production of polyclonal antibodies, hostanimals such as those described above, may be immunized by injectionwith endostatin gene product supplemented with adjuvants as alsodescribed above.

Monoclonal antibodies, which are homogeneous populations of antibodiesto a particular antigen, may be obtained by any technique that providesfor the production of antibody molecules by continuous cell lines inculture. These include, but are not limited to, the hybridoma techniqueof Kohler and Milstein, (1975, Nature 256, 495-497; and U.S. Pat. No.4,376,110), the human B-cell hybridoma technique (Kosbor et al., 1983,Immunology Today 4, 72; Cole et al., 1983, Proc. Natl. Acad. Sci. USA80, 2026-2030), and the EBV-hybridoma technique (Cole et al., 1985,Monoclonal Antibodies And Cancer Therapy, Alan R. Liss, Inc., pp.77-96). Such antibodies may be of any immunoglobulin class includingIgG, IgM, IgE, IgA, IgD and any subclass thereof. The hybridomaproducing the mAb of this invention may be cultivated in vitro or invivo. Production of high titers of mAbs in vivo makes this the presentlypreferred method of production.

Additionally, recombinant antibodies, such as chimeric and humanizedmonoclonal antibodies, comprising both human and non-human portions,which can be made using standard recombinant DNA techniques, are withinthe scope of the invention. A chimeric antibody is a molecule in whichdifferent portions are derived from different animal species, such asthose having a variable region derived from a murine mAb and a humanimmunoglobulin constant region. (See, e.g., Cabilly et al., U.S. Pat.No. 4,816,567; and Boss et al., U.S. Pat. No. 4,816397, which areincorporated herein by reference in their entirety.) Humanizedantibodies are antibody molecules from non-human species having one ormore complementarity determining regions (CDRs) from the non-humanspecies and a framework region from a human immunoglobulin molecule.(See, e.g., Queen, U.S. Pat. No. 5,585,089, which is incorporated hereinby reference in its entirety.) Such chimeric and humanized monoclonalantibodies can be produced by recombinant DNA techniques known in theart, for example using methods described in PCT Publication No. WO87/02671; European Patent Application 184,187; European PatentApplication 171,496; European Patent Application 173,494; PCTPublication No. WO 86/01533; U.S. Pat. No. 4,816,567; European PatentApplication 125,023; Better et al. (1988) Science 240:1041-1043; Liu etal. (1987) Proc. Natl. Acad. Sci. USA 84:3439-3443; Liu et al. (1987) J.Immunol 139:3521-3526; Sun et al. (1987) Proc. Natl. Acad. Sci. USA84:214-218; Nishimura et al. (1987) Canc. Res. 47:999-1005; Wood et al.(1985) Nature 314:446-449; and Shaw et al. (1988) J. Natl. Cancer Inst.80:1553-1559); Morrison (1985) Science 229:1202-1207; Oi et al. (1986)Bio/Techniques 4:214; U.S. Pat. No. 5,225,539; Jones et al. (I1986)Nature 321:552-525; Verhoeyan et al. (1988) Science 239:1534; andBeidler et al. (1988) J. Immunol. 141:4053-4060.

Completely human antibodies are particularly desirable for therapeutictreatment of human patients. Such antibodies can be produced, forexample, using transgenic mice which are incapable of expressingendogenous immunoglobulin heavy and light chains genes, but which canexpress human heavy and light chain genes. The transgenic mice areimmunized in the normal fashion with a selected antigen, e.g., all or aportion of a polypeptide of the invention. Monoclonal antibodiesdirected against the antigen can be obtained using conventionalhybridoma technology. The human immunoglobulin transgenes harbored bythe transgenic mice rearrange during B cell differentiation, andsubsequently undergo class switching and somatic mutation. Thus, usingsuch a technique, it is possible to produce therapeutically useful IgG,IgA and IgE antibodies. For an overview of this technology for producinghuman-antibodies,'see Lonberg and Huszar (1995, Int. Rev. Immunol.13:65-93). For a detailed discussion of this technology for producinghuman antibodies and human monoclonal antibodies and protocols forproducing such antibodies, see, e.g., U.S. Pat. No. 5,625,126; U.S. Pat.No. 5,633,425; U.S. Pat. No. 5,569,825; U.S. Pat. No. 5,661,016; andU.S. Pat. No. 5,545,806. In addition, companies such as Abgenix, Inc.(Fremont, Calif.), can be engaged to provide human antibodies directedagainst a selected antigen using technology similar to that describedabove.

Completely human antibodies which recognize a selected epitope can begenerated using a technique referred to as “guided selection.” In thisapproach a selected non-human monoclonal antibody, e.g., a mouseantibody, is used to guide the selection of a completely human antibodyrecognizing the same epitope. (Jespers et al. (1994) Bio/technology12:899-903).

Alternatively, techniques described for the production of single chainantibodies (U.S. Pat. No. 4,946,778; Bird, 1988, Science 242, 423-426;Huston, et al., 1988, Proc. Natl. Acad. Sci. USA 85, 5879-5883; andWard, et al., 1989, Nature 334, 544-546) can be adapted to producesingle-chain antibodies against endostatin gene products. Single chainantibodies are formed by linking the heavy and light chain fragments ofthe Fv region via an amino acid bridge, resulting in a single chainpolypeptide.

Antibody fragments that recognize specific epitopes may be generated byknown techniques. For example, such fragments include, but are notlimited to, the F(ab′)2 fragments, which can be produced by pepsindigestion of the antibody molecule and the Fab fragments, which can begenerated by reducing the disulfide bridges of the F(ab′)2 fragments.Alternatively, Fab expression libraries may be constructed (Huse, etal., 1989, Science, 246, 1275-1281) to allow rapid and easyidentification of monoclonal Fab fragments with the desired specificity.

Described herein are various applications of endostatin gene sequences,endostatin gene products, including peptide fragments and fusionproteins thereof, and of antibodies directed against endostatin geneproducts and peptide fragments thereof. Such applications include, forexample, prognostic and diagnostic evaluation of an angiogenesis-relateddisorder, e.g., cancer, and the identification of subjects with apredisposition to such disorders, as described, below, in Section.Additionally, such applications include methods for the identificationof compounds that modulate the expression of an endostatin gene and/orthe synthesis or activity of an endostatin gene product, as describedbelow, and for the treatment of an angiogenesis-related disorder, e.g.cancer, as described, below.

In addition, endostatin gene sequences and gene products, includingpeptide fragments and fusion proteins thereof, and antibodies directedagainst endostatin gene products and peptide fragments thereof, haveapplications for purposes independent of the role endostatin may have inangiogenesis-related disorders and processes. For example, endostatingene products, including peptide fragments, as well asendostatin-specific antibodies, can be used for construction of fusionproteins to facilitate recovery, detection, or localization of anotherprotein of interest. In addition, endostatin genes and gene products canbe used for genetic mapping, i.e., refining the genetic map. Forexample, antibodies specific to endostatin can be used as probes todetect expression of human endostatin in somatic cell hybrids containinghuman chromosomes, or portions of human chromosomes. Suchendostatin-specific antibodies can be used to identify cells thatcontain the endostatin chromosomal region. This method can be used, forexample, to localize a gene or trait of interest to this region of thechromosome.

Endostatin gene sequences and gene products can be used for mapping andrefining a chromosomal map. The endostatin sequence can be used todevelop new genetic markers to further refine chromosomal intervals thatare associated with various angiogenesis-related disorders, including,but not limited to, cancer. As will be apparent to the skilled artisan,nucleic acid sequences within a genetic interval associated with adisease can be scanned for new markers, such as microsatellites.Microsatellites, also known as simple-sequence repeats (SSRs), arehypervariable tandem-sequence repeats consisting of di-, tri-, ortetranucleotide repeats of 1-5 nucleotides. Such microsatellites makeexcellent genetic markers for linkage studies since they are distributedubiquitously throughout the human genome, are highly variable in repeatlength, and tend to be highly polymorphic. Relatively commonmicrosatellites (e.g., (CA)n dinucleotide repeats) occur approximatelyevery 300-500 kb. In addition to microsatellite repeats, the region canbe scanned for other types of polymorphic sites useful for fine mapping,such as minisatellites (9-64 nucleotide repeats), restriction fragmentlength polymorphisms (RFLPs), and single nucleotide polymorphisms, whichoccur much less frequently. Once a polymorphic site is identified in anew sequence, PCR primers that flank the polymorphic site can besynthesized and used to amplify the microsatellite or other polymorphicsite. The length of the repeat can then determined by resolving the PCRproduct on a polyacrylamide sequencing gel. Genomic DNA from humanpopulations can then be analyzed for the simple-sequence lengthpolymorphisms (SSLPs) to determine the frequency and variability of therepeat. Once a high quality SSLP is found, linkage analysis can beperformed on an affected population to determine whether anangiogenesis-related disorder, such as cancer, is linked to the newmarker. Other techniques, such as Southern blot hybridization andligase-chain reaction (LCR), can be used in addition to, or inconjunction with, PCR-based methods to analyze polymorphisms in genomicpopulations (see, Current Protocols in Human Genetics, Dracopoli et al.(eds.) John Wiley & Sons, 1998).

In another embodiment, an endostatin gene, protein or a fragment ordomain thereof, can be used for construction of fusion proteins.Finally, endostatin nucleic acids and gene products have generic uses,such as supplemental sources of nucleic acids, proteins and amino acidsfor food additives or cosmetic products.

Portions or fragments of the cDNA sequences identified herein (and thecorresponding complete gene sequences) can be used in numerous ways aspolynucleotide reagents. For example, these sequences can be used to:(i) screen for endostatin gene-specific mutations or polymorphisms, (ii)map their respective genes on a chromosome and, thus, locate generegions associated with genetic disease; (iii) identify an individualorganism from a minute biological sample (tissue typing); and (iv) aidin forensic identification of a biological sample. These applicationsare described in the subsections below.

A variety of methods can be employed to screen for the presence ofendostatin gene-specific mutations or polymorphisms (includingpolymorphisms flanking an endostatin gene, e.g., ones that cosegregatewith a particular endostatin allele) and to detect and/or assay levelsof endostatin nucleic acid sequences.

Mutations or polymorphisms within or flanking the endostatin gene can bedetected by utilizing a number of techniques. Nucleic acid from anynucleated cell, or any cell that expresses the endostatin gene ofinterest, can be used as the starting point for such assay techniques,and may be isolated according to standard nucleic acid preparationprocedures that are well known to those of skill in the art.

Endostatin nucleic acid sequences may be used in hybridization oramplification assays of biological samples to detect abnormalitiesinvolving endostatin gene structure, including point mutations,insertions, deletions, inversions, translocations and chromosomalrearrangements. Such assays may include, but are not limited to,Southern analyses, single-stranded conformational polymorphism analyses(SSCP), and PCR analyses.

Diagnostic methods for the detection of endostatin gene-specificmutations or polymorphisms can involve, for example, contacting andincubating nucleic acids obtained from a sample, e.g., derived from apatient sample or other appropriate cellular source with one or morelabeled nucleic acid reagents including recombinant DNA molecules,cloned genes or degenerate variants thereof, such as described, above,under conditions favorable for the specific annealing of these reagentsto their complementary sequences within or flanking the endostatin gene.The diagnostic methods of the present invention further encompasscontacting and incubating nucleic acids for the detection of singlenucleotide mutations or polymorphisms of the endostatin gene.Preferably, these nucleic acid reagent sequences within the endostatingene, or chromosome nucleotide sequences flanking the endostatin geneare 15 to 30 nucleotides in length.

After incubation, all non-annealed nucleic acids are removed from thenucleic acid:endostatin molecule hybrid. The presence of nucleic acidsthat have hybridized, if any such molecules exist, is then detected.Using such a detection scheme, the nucleic acid from the cell type ortissue of interest can be immobilized, for example, to a solid supportsuch as a membrane, or a plastic surface such as that on a microtiterplate or polystyrene beads. In this case, after incubation,non-annealed, labeled nucleic acid reagents of the type described above,are easily removed. Detection of the remaining, annealed, labeledendostatin nucleic acid reagents is accomplished using standardtechniques well-known to those in the art. The endostatin gene sequencesto which the nucleic acid reagents have annealed can be compared to theannealing pattern expected from a normal endostatin gene sequence inorder to determine whether an endostatin gene mutation is present.

In a preferred embodiment, endostatin mutations or polymorphisms can bedetected by using a microassay of endostatin nucleic acid sequencesimmobilized to a substrate or “gene chip” (see, e.g. Cronin et al.,1996, Human Mutation 7:244-255).

Alternative diagnostic methods for the detection of endostatingene-specific nucleic acid molecules (or endostatin flanking sequences),in patient samples or other appropriate cell sources, may involve theiramplification, e.g., by PCR (the experimental embodiment set forth inMullis, 1987, U.S. Pat. No. 4,683,202), followed by the analysis of theamplified molecules using techniques well known to those of skill in theart, such as, for example, those listed above. The resulting amplifiedsequences can be compared to those that would be expected if the nucleicacid being amplified contained only normal copies of the endostatin genein order to determine whether an endostatin gene mutation orpolymorphism in linkage disequilibrium with a disease-causing endostatinallele exists.

Additionally, well-known genotyping techniques can be performed toidentify individual organisms carrying endostatin gene mutations. Suchtechniques include, for example, the use of restriction fragment lengthpolymorphisms (RFLPs), which involve sequence variations in one of therecognition sites for the specific restriction enzyme used.

Further, improved methods for analyzing DNA polymorphisms, which can beutilized for the identification of endostatin gene-specific mutations,have been described that capitalize on the presence of variable numbersof short, tandemly repeated DNA sequences between the restriction enzymesites. For example, Weber (U.S. Pat. No. 5,075,217) describes a DNAmarker based on length polymorphisms in blocks of (dC-dA)n-(dG-dT)nshort tandem repeats. The average separation of (dC-dA)n-(dG-dT)n blocksis estimated to be 30,000-60,000 bp. Markers that are so closely spacedexhibit a high frequency co-inheritance, and are extremely useful in theidentification of genetic mutations, such as, for example, mutationswithin the endostatin gene, and the diagnosis of diseases and disordersrelated to endostatin mutations.

Also, Caskey et al. (U.S. Pat. No. 5,364,759) describe a DNA profilingassay for detecting short tri and tetra nucleotide repeat sequences. Theprocess includes extracting the DNA of interest, such as the endostatingene, amplifying the extracted DNA, and labeling the repeat sequences toform a genotypic map of the individual organism's DNA.

Other methods well known in the art may be used to identify singlenucleotide polymorphisms (SNPs), including biallelic SNPs or biallelicmarkers which have two alleles, both of which are present at a fairlyhigh frequency in a population. Conventional techniques for detectingSNPs include, e.g., conventional dot blot analysis, SSCP analysis (see,e.g., Orita et al., 1989, Proc. Natl. Acad. Sci. USA 86:2766-2770),denaturing gradient gel electrophoresis (DGGE), heteroduplex analysis,mismatch cleavage detection, and other routine techniques well known inthe art (see, e.g., Sheffield et al., 1989, Proc. Natl. Acad. Sci.86:5855-5892; Grompe, 1993, Nature Genetics 5:111-117). Alternative,preferred methods of detecting and mapping SNPs involve microsequencingtechniques wherein an SNP site in a target DNA is detecting by a singlenucleotide primer extension reaction (see, e.g., Goelet et al., PCTPublication No. WO92/15712; Mundy, U.S. Pat. No. 4,656,127; Vary andDiamond, U.S. Pat. No. 4,851,331; Cohen et al., PCT Publication No.WO91/02087; Chee et al., PCT Publication No. WO95/11995; Landegren etal., 1988, Science 241:1077-1080; Nicerson et al., 1990, Proc. Natl.Acad. Sci. U.S.A. 87:8923-8927; Pastinen et al., 1997, Genome Res.7:606-614; Pastinen et al., 1996, Clin. Chem. 42:1391-1397; Jalanko etal., 1992, Clin. Chem. 38:39-43; Shumaker et al., 1996, Hum. Mutation7:346-354; Caskey et al., PCT Publication No. WO 95/00669).

The level of endostatin expression also can be determined by firstassaying for the level of gene expression. For example, RNA from a celltype or tissue known, or suspected, to express the endostatin gene, suchas liver, may be isolated and tested utilizing hybridization or PCRtechniques such as are described, above. The isolated cells can bederived from cell culture or from a subject. The analysis of cells takenfrom culture may be a necessary step in the assessment of cells to beused as part of a cell-based gene therapy technique or, alternatively,to test the effect of compounds on the expression of the endostatingene.

In one embodiment of such a detection scheme, a cDNA molecule issynthesized from an RNA molecule of interest (e.g., by reversetranscription of the RNA molecule into cDNA). A sequence within the cDNAis then used as the template for a nucleic acid amplification reaction,such as a PCR amplification reaction, or the like. The nucleic acidreagents used as synthesis initiation reagents (e.g., primers) in thereverse transcription and nucleic acid amplification steps of thismethod are chosen from among the endostatin gene nucleic acid reagentsdescribed above. The preferred lengths of such nucleic acid reagents areat least 9-30 nucleotides. For detection of the amplified product, thenucleic acid amplification may be performed using radioactively ornon-radioactively labeled nucleotides. Alternatively, enough amplifiedproduct may be made such that the product may be visualized by standardethidium bromide staining or by utilizing any other suitable nucleicacid staining method.

Additionally, it is possible to perform such endostatin gene expressionassays in situ, i.e., directly upon tissue sections (fixed and/orfrozen) of subject tissue obtained from biopsies or resections, suchthat no nucleic acid purification is necessary. Nucleic acid reagentssuch as those described above, may be used as probes and/or primers forsuch in situ procedures (see, for example, Nuovo, G. J., 1992, “PCR InSitu Hybridization: Protocols And Applications”, Raven Press, NY).

Alternatively, if a sufficient quantity of the appropriate cells can beobtained, standard Northern analysis can be performed to determine thelevel of mRNA expression of the endostatin gene.

Once the sequence (or a portion of the sequence) of a gene has beenisolated, this sequence can be used to map the location of the gene on achromosome. Accordingly, nucleic acid molecules described herein, orfragments thereof, can be used to map the location of the correspondinggenes on a chromosome. The mapping of the sequences to chromosomes is animportant first step in correlating these sequences with genesassociated with disease.

Briefly, genes can be mapped to chromosomes by preparing PCR primers(preferably 15-25 bp in length) from the sequence of a gene of theinvention. Computer analysis of the sequence of a gene of the inventioncan be used to rapidly select primers that do not span more than oneexon in the genomic DNA, thus simplifying the amplification process.These primers can then be used for PCR screening of somatic cell hybridscontaining individual human chromosomes. Only those hybrids containingthe human gene corresponding to the gene sequences will yield anamplified fragment. For a review of this technique, see D'Eustachio etal., 1983, Science, 220:919-924.

PCR mapping of somatic cell hybrids is a rapid procedure for assigning aparticular sequence to a particular chromosome. Three or more sequencescan be assigned per day using a single thermal cycler. Using the nucleicacid sequences of the invention to design oligonucleotide primers,sublocalization can be achieved with panels of fragments from specificchromosomes. Other mapping strategies which can similarly be used to mapa gene to its chromosome include in situ hybridization (described in Fanet al., 1990, Proc. Natl. Acad. Sci. USA 87:6223-27), pre-screening withlabeled flow-sorted chromosomes (Popp S, et al., 1993, Hum Genet.,92(6):527-32) and pre-selection by hybridization to chromosome specificcDNA libraries. Fluorescence in situ hybridization (FISH) of a DNAsequence to a metaphase chromosomal spread can further be used toprovide a precise chromosomal location in one step. (For a review ofthis technique, see Verma et al., Human Chromosomes: A Manual of BasicTechniques, Pergamon Press, New York, 1988.)

Reagents for chromosome mapping can be used individually to mark asingle chromosome or a single site on that chromosome, or panels ofreagents can be used for marking multiple sites and/or multiplechromosomes. Reagents corresponding to noncoding regions of the genesactually are preferred for mapping purposes. Coding sequences are morelikely to be conserved within gene families, thus increasing the chanceof cross hybridizations during chromosomal mapping.

Once a sequence has been mapped to a precise chromosomal location, thephysical position of the sequence on the chromosome can be correlatedwith genetic map data. (Such data are found, for example, in V.McKusick, Mendelian Inheritance in Man, available on-line through JohnsHopkins University Welch Medical Library). The relationship betweengenes and disease, mapped to the same chromosomal region, can then beidentified through linkage analysis (co-inheritance of physicallyadjacent genes), described in, e.g., Egeland et al.,1987, Nature325:783-787.

Moreover, differences in the DNA sequences between individual organismsaffected and unaffected with a disease associated with a gene of theinvention can be determined. If a mutation is observed in some or all ofthe affected individual organisms but not in any unaffected individualorganisms, then the mutation is likely to be the causative agent of theparticular disease. Comparison of affected and unaffected individualorganisms generally involves first looking for structural alterations inthe chromosomes such as deletions or translocations that are visiblefrom chromosome spreads or detectable using PCR based on that DNAsequence. Ultimately, complete sequencing of genes from severalindividual organisms can be performed to confirm the presence of amutation and to distinguish mutations from polymorphisms.

The nucleic acid sequences of the present invention also can be used toidentify individual organisms from minute biological samples. The UnitedStates military, for example, is considering the use of restrictionfragment length polymorphism (RFLP) for identification of its personnel.In this technique, an individual organism's genomic DNA is digested withone or more restriction enzymes, and probed on a Southern blot to yieldunique bands for identification. This method does not suffer from thecurrent limitations of “Dog Tags” which can be lost, switched, orstolen, making positive identification difficult. The sequences of thepresent invention are useful as additional DNA markers for RFLP(described in U.S. Pat. No. 5,272,057).

Furthermore, the sequences of the present invention can be used toprovide an alternative technique which determines the actualbase-by-base DNA sequence of selected portions of an individualorganism's genome. Thus, the nucleic acid sequences described herein canbe used to prepare two PCR primers from the 5′ and 3′ ends of thesequences. These primers can then be used to amplify an individualorganism's DNA and subsequently sequence it.

Panels of corresponding DNA sequences from individual organisms,prepared in this manner, can provide unique individual identifications,as each individual organism will have a unique set of such DNA sequencesdue to allelic differences. The sequences of the present invention canbe used to obtain such identification sequences from individualorganisms and from tissue. The nucleic acid sequences of the inventionuniquely represent portions of the human genome. Allelic variationoccurs to some degree in the coding regions of these sequences, and to agreater degree in the noncoding regions. It is estimated that allelicvariation between individual humans occurs with a frequency of aboutonce per each 500 bases. Each of the sequences described herein can, tosome degree, be used as a standard against which DNA from an individualorganism can be compared for identification purposes. Because greaternumbers of polymorphisms occur in the noncoding regions, fewer sequencesare necessary to differentiate individuals.

If a panel of reagents from the nucleic acid sequences described hereinis used to generate a unique identification database for an individual,those same reagents can later be used to identify tissue from thatindividual. Using the unique identification database, positiveidentification of the individual organism, living or dead, can be madefrom extremely small tissue samples.

DNA-based identification techniques can also be used in forensicbiology. Forensic biology is a scientific field employing genetic typingof biological evidence found at a crime scene as a means for positivelyidentifying, for example, a perpetrator of a crime. To make such anidentification, PCR technology can be used to amplify DNA sequencestaken from very small biological samples such as tissues, e.g., hair orskin, or body fluids, e.g., blood, saliva, or semen found at a crimescene. The amplified sequence can then be compared to a standard,thereby allowing identification of the origin of the biological sample.

The sequences of the present invention can be used to providepolynucleotide reagents, e.g., PCR primers, targeted to specific loci inthe human genome, which can enhance the reliability of DNA-basedforensic identifications by, for example, providing another“identification marker” (i.e., another DNA sequence that is unique to aparticular individual organism). As mentioned above, actual basesequence information can be used for identification as an accuratealternative to patterns formed by restriction enzyme generatedfragments. Sequences targeted to noncoding regions are particularlyappropriate for this use as greater numbers of polymorphisms occur inthe noncoding regions, making it easier to differentiate individualorganisms using this technique. Examples of polynucleotide reagentsinclude the nucleic acid sequences of the invention or portions thereof,e.g., fragments derived from noncoding regions having a length of atleast 20 or 30 bases.

The nucleic acid sequences described herein can further be used toprovide polynucleotide reagents, e.g., labeled or labelable probes whichcan be used in, for example, an in situ hybridization technique, toidentify a specific tissue, e.g., liver tissue. This can be very usefulin cases where a forensic pathologist is presented with a tissue ofunknown origin. Panels of such probes can be used to identify tissue byspecies and/or by organ type.

Antibodies directed against unimpaired or mutant endostatin geneproducts or conserved variants or peptide fragments thereof, which arediscussed, above, may also be used as diagnostics and prognostics foran-angiogenesis-related disorder, e.g., cancer, as described herein.Such methods may be used to detect abnormalities in the level ofendostatin gene product synthesis or expression, or abnormalities in thestructure, temporal expression, and/or physical location of endostatingene product. The antibodies and immunoassay methods described belowhave, for example, important in vitro applications in purifyingendostatin gene products and in assessing the efficacy of treatments forangiogenesis-related disorders, e.g., cancer. Antibodies, or fragmentsof antibodies, such as those described below, may be used to screenpotentially therapeutic compounds in vitro to determine their effects onendostatin gene expression and endostatin peptide production. Thecompounds that have beneficial effects on an angiogenesis-relateddisorder, e.g., cancer, can be identified, and a therapeuticallyeffective dose determined.

In vitro immunoassays may also be used, for example, to assess theefficacy of cell-based gene therapy for an angiogenesis-relateddisorder, e.g., cancer. Antibodies directed against endostatin peptidesmay be used in vitro to determine, for example, the level of endostatingene expression achieved in cells genetically engineered to produceendostatin peptides. In the case of intracellular endostatin geneproducts, such an assessment is done, preferably, using cell lysates orextracts. Such analysis allows for a determination of the number oftransformed cells necessary to achieve therapeutic efficacy in vivo, aswell as optimization of the gene replacement protocol.

The tissue or cell type to be analyzed will generally include those thatare known, or suspected, to express the endostatin gene. The proteinisolation methods employed herein may, for example, be such as thosedescribed in Harlow and Lane (1988, “Antibodies: A Laboratory Manual”,Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y.). Theisolated cells can be derived from cell culture or from a subject. Theanalysis of cells taken from culture may be a necessary step in theassessment of cells to be used as part of a cell-based gene therapytechnique or, alternatively, to test the effect of compounds on theexpression of the endostatin gene.

Preferred diagnostic methods for the detection of endostatin geneproducts or conserved variants or peptide fragments thereof, mayinvolve, for example, immunoassays wherein the endostatin gene productsor conserved variants or peptide fragments are detected by theirinteraction with an anti-endostatin gene product-specific antibody.

For example, antibodies, or fragments of antibodies, such as thosedescribed, above, useful in the present invention may be used toquantitatively or qualitatively detect the presence of endostatin geneproducts or conserved variants or peptide fragments thereof. This can beaccomplished, for example, by immunofluorescence techniques employing afluorescently labeled antibody (see below, this section) coupled withlight microscopic, flow cytometric, or fluorimetric detection. Suchtechniques are especially preferred for endostatin gene products thatare expressed on the cell surface.

The antibodies (or fragments thereof) useful in the present inventionmay, additionally, be employed histologically, as in immunofluorescenceor immunoelectron microscopy, for in situ detection of endostatin geneproducts or conserved variants or peptide fragments thereof. In situdetection may be accomplished by removing a histological specimen from asubject, and applying thereto a labeled antibody of the presentinvention. The antibody (or fragment) is preferably applied byoverlaying the labeled antibody (or fragment) onto a biological sample.Through the use of such a procedure, it is possible to determine notonly the presence of the endostatin gene product, or conserved variantsor peptide fragments, but also its distribution in the examined tissue.Using the present invention, those of ordinary skill will readilyperceive that any of a wide variety of histological methods (such asstaining procedures) can be modified in order to achieve such in situdetection.

Immunoassays for endostatin gene products or conserved variants orpeptide fragments thereof will typically comprise incubating a sample,such as a biological fluid, a tissue extract, freshly harvested cells,or lysates of cells, that have been incubated in cell culture, in thepresence of a detectably labeled antibody capable of identifyingendostatin gene products or conserved variants or peptide fragmentsthereof, and detecting the bound antibody by any of a number oftechniques well-known in the art.

The biological sample may be brought in contact with and immobilizedonto a solid phase support or carrier such as nitrocellulose, or othersolid support that is capable of immobilizing cells, cell particles orsoluble proteins. The support may then be washed with suitable buffersfollowed by treatment with the detectably labeled endostatin genespecific antibody. The solid phase support may then be washed with thebuffer a second time to remove unbound antibody. The amount of boundlabel on solid support may then be detected by conventional means.

By “solid phase support or carrier” is intended any support capable ofbinding an antigen or an antibody. Well-known supports or carriersinclude glass, polystyrene, polypropylene, polyethylene, dextran, nylon,amylases, natural and modified celluloses, polyacrylamides, gabbros, andmagnetite. The nature of the carrier can be either soluble to someextent or insoluble for the purposes of the present invention. Thesupport material may have virtually any possible structuralconfiguration so long as the coupled molecule is capable of binding toan antigen or antibody. Thus, the support configuration may bespherical, as in a bead, or cylindrical, as in the inside surface of atest tube, or the external surface of a rod. Alternatively, the surfacemay be flat such as a sheet, test strip, etc. Preferred supports includepolystyrene beads. Those skilled in the art will know many othersuitable carriers for binding antibody or antigen, or will be able toascertain the same by use of routine experimentation.

The binding activity of a given lot of anti-endostatin gene productantibody may be determined according to well known methods. Thoseskilled in the art will be able to determine operative and optimal assayconditions for each determination by employing routine experimentation.

One of the ways in which the endostatin gene peptide-specific antibodycan be detectably labeled is by linking the same to an enzyme and use inan enzyme immunoassay (EIA) (Voller, A., “The Enzyme LinkedImmunosorbent Assay (ELISA)”, 1978, Diagnostic Horizons 2, 1-7,Microbiological Associates Quarterly Publication, Walkersville, Md.);Voller, A. et al., 1978, J. Clin. Pathol. 31, 507-520; Butler, J. E.,1981, Meth. Enzymol. 73, 482-523; Maggio, E. (ed.), 1980, EnzymeImmunoassay, CRC Press, Boca Raton, Fla.,; Ishikawa, E. et al., (eds.),1981, Enzyme Immunoassay, Kgaku Shoin, Tokyo). The enzyme which is boundto the antibody will react with an appropriate substrate, preferably achromogenic substrate, in such a manner as to produce a chemical moietythat can be detected, for example, by spectrophotometric, fluorimetricor by visual means. Enzymes that can be used to detectably label theantibody include, but are not limited to, malate dehydrogenase,staphylococcal nuclease, delta-5-steroid isomerase, yeast alcoholdehydrogenase, β-glycerophosphate, dehydrogenase, triose phosphateisomerase, horseradish peroxidase, alkaline phosphatase, asparaginase,glucose oxidase, β-galactosidase, ribonuclease, urease, catalase,glucose-6-phosphate dehydrogenase, glucoamylase andacetylcholinesterase. The detection can be accomplished by colorimetricmethods that employ a chromogenic substrate for the enzyme. Detectionmay also be accomplished by visual comparison of the extent of enzymaticreaction of a substrate in comparison with similarly prepared standards.

Detection also may be accomplished using any of a variety of otherimmunoassays. For example, by radioactively labeling the antibodies orantibody fragments, it is possible to detect endostatin gene peptidesthrough the use of a radioimmunoassay (RIA) (see, for example,Weintraub, B., Principles of Radioimmunoassays, Seventh Training Courseon Radioligand Assay Techniques, The Endocrine Society, March, 1986).The radioactive isotope can be detected by such means as the use of agamma counter or a scintillation counter or by autoradiography.

It is also possible to label the antibody with a fluorescent compound.When the fluorescently labeled antibody is exposed to light of theproper wave length, its presence can then be detected due tofluorescence. Among the most commonly used fluorescent labelingcompounds are fluorescein isothiocyanate, rhodamine, phycoerythrin,phycocyanin, allophycocyanin, o-phthaldehyde and fluorescamine.

The antibody can also be detectably labeled using fluorescence emittingmetals such as 152Eu, or others of the lanthanide series. These metalscan be attached to the antibody using such metal chelating groups asdiethylenetriaminepentacetic acid (DTPA) or ethylenediaminetetraaceticacid (EDTA).

The antibody also can be detectably labeled by coupling it to achemiluminescent compound. The presence of the chemiluminescent-taggedantibody is then determined by detecting the presence of luminescencethat arises during the course of a chemical reaction. Examples ofparticularly useful chemiluminescent labeling compounds are luminol,isoluminol, theromatic acridinium ester, imidazole, acridinium salt andoxalate ester.

Likewise, a bioluminescent compound may be used to label the antibody ofthe present invention. Bioluminescence is a type of chemiluminescencefound in biological systems in which a catalytic protein increases theefficiency of the chemiluminescent reaction. The presence of abioluminescent protein is determined by detecting the presence ofluminescence. Important bioluminescent compounds for purposes oflabeling are luciferin, luciferase and aequorin.

The following assays are designed to identify compounds that bind to anendostatin gene product, proteins, e.g., intracellular proteins orportions of proteins that interact with an endostatin gene product,compounds that interfere with the interaction of an endostatin geneproduct with intracellular proteins and compounds that modulate theactivity of an endostatin gene (i.e., modulate the level of endostatingene expression and/or modulate the level of endostatin gene productactivity). Assays may additionally be utilized that identify compoundsthat bind to endostatin gene regulatory sequences (e.g., promotersequences; see e.g., Platt, 1994, J. Biol. Chem. 269, 28558-28562), andthat may modulate the level of endostatin gene expression. Compounds mayinclude, but are not limited to, small organic molecules, such as onesthat are able to cross the blood-brain barrier, gain entry into anappropriate cell and affect expression of the endostatin gene or someother gene involved in an endostatin regulatory pathway, orintracellular proteins.

Methods for the identification of such intracellular proteins aredescribed, below. Such intracellular proteins may be involved in thecontrol and/or regulation of mood. Further, among these compounds arecompounds that affect the level of endostatin gene expression and/orendostatin gene product activity and that can be used in the therapeutictreatment of endostatin disorders, e.g., cancer, as described, below.

Compounds may include, but are not limited to, peptides such as, forexample, soluble peptides, including, but not limited to, Ig-tailedfusion peptides, and members of random peptide libraries; (see, e.g.,Lam, et al., 1991, Nature 354, 82-84; Houghten, et al., 1991, Nature354, 84-86), and combinatorial chemistry-derived molecular library madeof D- and/or L-configuration amino acids, phosphopeptides (including,but not limited to members of random or partially degenerate, directedphosphopeptide libraries; see, e.g., Songyang, et al., 1993, Cell 72,767-778), antibodies (including, but not limited to, polyclonal,monoclonal, human, humanized, anti-idiotypic, chimerc or single chainantibodies, and FAb, F(ab □)2 and FAb expression library fragments, andepitope-binding fragments thereof), and small organic or inorganicmolecules.

Such compounds may also comprise compounds, in particular drugs ormembers of classes or families of drugs, known to ameliorate orexacerbate the symptoms of an angiogenesis-related disorder. Suchcompounds include, but are not limited to, angiogenesis inhibitors:metalloproteinase inhibitors, FGF and VEGF receptor inhibitors, COX-2inhibitors, INF, IL-12, Taxol, vinblastine, thalidomide etc. Preferablysuch compounds are utilized in a manner (e.g., different dosage, mode ofadministration, and/or co-administration with one or more additionalcompounds) that differs from the manner in which such compounds havebeen administered previously.

Compounds identified via assays such as those described herein may beUseful, for example, in elaborating the biological function of theendostatin gene product, and for ameliorating angiogenesis-relateddisorders, e.g., cancer. For example, compounds identified via suchtechniques can provide lead compounds to be tested for an ability tomodulate an endostatin-mediated process and/or to ameliorate symptoms ofa angiogenesis-related disorder. Assays for testing the effectiveness ofcompounds, identified by, for example, techniques such as thosedescribed above, are discussed, below.

In vitro systems may be designed to identify compounds that bindendostatin gene products of the invention, such as, for example,endostatin polypeptides. Compounds identified may be useful, forexample, in modulating the activity of unimpaired and/or mutantendostatin gene products, may be useful in elucidating the biologicalfunction of the endostatin gene product, may be utilized in screens foridentifying compounds that disrupt normal endostatin gene productinteractions, or may in themselves disrupt such interactions, and canprovide lead compounds to be further tested for an ability to modulatean endostatin-mediated process and/or to ameliorate symptoms of anangiogenesis-related disorder.

The principle of the assays used to identify compounds that bind toendostatin gene products involves preparing a reaction mixture of theendostatin gene product and the test compound under conditions and for atime sufficient to allow the two components to interact and bind, thusforming a complex that can be removed and/or detected in the reactionmixture. These assays can be conducted in a variety of ways. Forexample, one method to conduct such an assay would involve anchoring anendostatin gene product or the test substance onto a solid phase anddetecting endostatin gene product/test compound complexes anchored onthe solid phase at the end of the reaction. In one embodiment of such amethod, the endostatin gene product may be anchored onto a solidsurface, and the test compound, which is not anchored, may be labeled,either directly or indirectly.

In practice, microtiter plates may conveniently be utilized as the solidphase. The anchored component may be immobilized by non-covalent orcovalent attachments. Non-covalent attachment may be accomplished bysimply coating the solid surface with a solution of the protein anddrying. Alternatively, an immobilized antibody, preferably a monoclonalantibody, specific for the protein to be immobilized may be used toanchor the protein to the solid surface. The surfaces may be prepared inadvance and stored.

In order to conduct the assay, the non-immobilized component is added tothe coated surface containing the anchored component. After the reactionis complete, unreacted components are removed (e.g., by washing) underconditions such that any complexes formed will remain immobilized on thesolid surface. The detection of complexes anchored on the solid surfacecan be accomplished in a number of ways. Where the previouslynon-immobilized component is pre-labeled, the detection of labelimmobilized on the surface indicates that complexes were formed. Wherethe previously non-immobilized component is not pre-labeled, an indirectlabel can be used to detect complexes anchored on the surface; e.g.,using a labeled antibody specific for the previously non-immobilizedcomponent (the antibody, in turn, may be directly labeled or indirectlylabeled with a labeled anti-Ig antibody).

Alternatively, a reaction can be conducted in a liquid phase, thereaction products separated from unreacted components, and complexesdetected; e.g., using an immobilized antibody specific for endostatingene product or the test compound to anchor any complexes formed insolution, and a labeled antibody specific for the other component of thepossible complex to detect anchored complexes.

Any method suitable for detecting protein-protein interactions may beemployed for identifying endostatin protein-protein interactions.

Among the traditional methods that may be employed areco-immunoprecipitation, cross-linking and co-purification throughgradients or chromatographic columns. Utilizing procedures such as theseallows for the identification of proteins, including intracellularproteins, that interact with endostatin gene products. Once isolated,such a protein can be identified and can be used in conjunction withstandard techniques, to identify proteins it interacts with. Forexample, at least a portion of the amino acid sequence of a protein thatinteracts with the endostatin gene product can be ascertained usingtechniques well known to those of skill in the art, such as via theEdman degradation technique (see, e.g., Creighton, 1983, “Proteins:Structures and Molecular Principles,” W.H. Freeman & Co., N.Y.,pp.3449). The amino acid sequence obtained may be used as a guide forthe generation of oligonucleotide mixtures that can be used to screenfor gene sequences encoding such proteins. Screening made beaccomplished, for example, by standard hybridization or PCR techniques.Techniques for the generation of oligonucleotide mixtures and thescreening are well-known. (See, e.g., Ausubel, supra, and 1990, “PCRProtocols: A Guide to Methods and Applications,” Innis, et al., eds.Academic Press, Inc., New York).

Additionally, methods may be employed that result in the simultaneousidentification of genes that encode a protein which interacts with anendostatin protein. These methods include, for example, probingexpression libraries with labeled endostatin protein, using endostatinprotein in a manner similar to the well known technique of antibodyprobing of gt11 libraries.

One method that detects protein interactions in vivo, the two-hybridsystem, is described in detail for illustration only and not by way oflimitation. One version of this system has been described (Chien, etal., 1991, Proc. Natl. Acad. Sci. USA, 88:9578-9582) and is commerciallyavailable from Clontech (Palo Alto, Calif.).

Briefly, utilizing such a system, plasmids are constructed that encodetwo hybrid proteins: one consists of the DNA-binding domain of atranscription activator protein fused to the endostatin gene product andthe other consists of the transcription activator protein's activationdomain fused to an unknown protein that is encoded by a cDNA that hasbeen recombined into this plasmid as part of a cDNA library. TheDNA-binding domain fusion plasmid and the cDNA library are transformedinto a strain of the yeast Saccharomyces cerevisiae that contains areporter gene (e.g., HBS or lacZ) whose regulatory region contains thetranscription activator's binding site. Either hybrid protein alonecannot activate transcription of the reporter gene: the DNA-bindingdomain hybrid cannot because it does not provide activation function andthe activation domain hybrid cannot because it cannot localize to theactivator's binding sites. Interaction of the two hybrid proteinsreconstitutes the functional activator protein and results in expressionof the reporter gene, which is detected by an assay for the reportergene product.

The two-hybrid system or related methodology may be used to screenactivation domain libraries for proteins that interact with the “bait”gene product. By way of example, and not by way of limitation,endostatin gene products may be used as the bait gene product. Totalgenomic or cDNA sequences are fused to the DNA encoding an activationdomain. This library and a plasmid encoding a hybrid of a baitendostatin gene product fused to the DNA-binding domain areco-transformed into a yeast reporter strain, and the resultingtransformants are screened for those that express the reporter gene. Forexample, and not by way of limitation, a bait endostatin gene sequence,such as the open reading frame of the endostatin gene, can be clonedinto a vector such that it is translationally fused to the DNA encodingthe DNA-binding domain of the GAL4 protein. These colonies are purifiedand the library plasmids responsible for reporter gene expression areisolated. DNA sequencing is then used to identify the proteins encodedby the library plasmids.

A cDNA library of the cell line from which proteins that interact withthe bait endostatin gene product can be made using methods routinelypracticed in the art. According to the particular system describedherein, for example, the cDNA fragments can be inserted into a vectorsuch that they are translationally fused to the transcriptionalactivation domain of GAL4. This library can be co-transformed along withthe bait endostatin gene-GAL4 fusion plasmid into a yeast strain thatcontains a lacZ gene driven by a promoter that contains GAL4 activationsequence. A cDNA encoded protein, fused to GAL4 transcriptionalactivation domain, that interacts with bait endostatin gene product willreconstitute an active GAL4 protein and thereby drive expression of theHIS3 gene. Colonies that express HIS3 can be detected by their growth onpetri dishes containing semi-solid agar based media lacking histidine.The cDNA can then be purified from these strains, and used to produceand isolate the bait endostatin gene-interacting protein usingtechniques routinely practiced in the art.

Endostatin gene products of the invention may, in vivo, interact withone or more macromolecules, including cellular or extracellularmacromolecules, such as proteins. Such macromolecules may include, butare not limited to, other proteins, such as cellular receptors, ornucleic acid molecules and those proteins identified via methods such asthose described, above. For example, the endostatin gene product mayinteract with a receptor as a peptide hormone or neuropeptide. Forpurposes of this discussion, the macromolecules are referred to hereinas “binding partners”. Compounds that disrupt endostatin binding in thisway may be useful in regulating the activity of the endostatin geneproduct, especially mutant endostatin gene products. For example, suchcompounds may interfere with the interaction of the endostatin geneproduct, with its receptor. Such compounds may include, but are notlimited to, molecules such as peptides, and the like, as described, forexample, above, which would be capable of gaining access to anendostatin gene product.

The basic principle of the assay systems used to identify compounds thatinterfere with the interaction between the endostatin gene product andits binding partner or partners involves preparing a reaction mixturecontaining the endostatin gene product, and the binding partner underconditions and for a time sufficient to allow the two to interact andbind, thus forming a complex. In order to test a compound for inhibitoryactivity, the reaction mixture is prepared in the presence and absenceof the test compound. The test compound may be initially included in thereaction mixture, or may be added at a time subsequent to the additionof the endostatin gene product and its binding partner. Control reactionmixtures are incubated without the test compound or with a placebo. Theformation of any complexes between the endostatin gene protein and thebinding partner is then detected. The formation of a complex in thecontrol reaction, but not in the reaction mixture containing the testcompound, indicates that the compound interferes with the interaction ofthe endostatin gene protein and the interactive binding partner.Additionally, complex formation within reaction mixtures containing thetest compound and normal endostatin gene protein may also be compared tocomplex formation within reaction mixtures containing the test compoundand a mutant endostatin gene protein. This comparison may be importantin those cases wherein it is desirable to identify compounds thatdisrupt interactions of mutant but not normal endostatin gene proteins.

The assay for compounds that interfere with the interaction of theendostatin gene products and binding partners can be conducted in aheterogeneous or homogeneous format. Heterogeneous assays involveanchoring either the endostatin gene product or the binding partner ontoa solid phase and detecting complexes anchored on the solid phase at theend of the reaction. In homogeneous assays, the entire reaction iscarried out in a liquid phase. In either approach, the order of additionof reactants can be varied to obtain different information about thecompounds being tested. For example, test compounds that interfere withthe interaction between the endostatin gene products and the bindingpartners, e.g., by competition, can be identified by conducting thereaction in the presence of the test substance; i.e., by adding the testsubstance to the reaction mixture prior to or simultaneously with theendostatin gene protein and interactive intracellular binding partner.Alternatively, test compounds that disrupt preformed complexes, e.g.,compounds with higher binding constants that displace one of thecomponents from the complex, can be tested by adding the test compoundto the reaction mixture after complexes have been formed. The variousformats are described briefly below.

In a heterogeneous assay system, either the endostatin gene product orthe interactive binding partner, is anchored onto a solid surface, whilethe non-anchored species is labeled, either directly or indirectly. Inpractice, microtiter plates are conveniently utilized. The anchoredspecies may be immobilized by non-covalent or covalent attachments.Non-covalent attachment may be accomplished simply by coating the solidsurface with a solution of the endostatin gene product or bindingpartner and drying. Alternatively, an immobilized antibody specific forthe species to be anchored may be used to anchor the species to thesolid surface. The surfaces may be prepared in advance and stored.

In order to conduct the assay, the partner of the immobilized species isexposed to the coated surface with or without the test compound. Afterthe reaction is complete, unreacted components are removed (e.g., bywashing) and any complexes formed will remain immobilized on the solidsurface. The detection of complexes anchored on the solid surface can beaccomplished in a number of ways. Where the non-immobilized species ispre-labeled, the detection of label immobilized on the surface indicatesthat complexes were formed. Where the non-immobilized species is notpre-labeled, an indirect label can be used to detect complexes anchoredon the surface; e.g., using a labeled antibody specific for theinitially non-immobilized species (the antibody, in turn, may bedirectly labeled or indirectly labeled with a labeled anti-Ig antibody).Depending upon the order of addition of reaction components, testcompounds that inhibit complex formation or that disrupt preformedcomplexes can be detected.

Alternatively, the reaction can be conducted in a liquid phase in thepresence or absence of the test compound, the reaction productsseparated from unreacted components, and complexes detected; e.g., usingan immobilized antibody specific for one of the binding components toanchor any complexes formed in solution, and a labeled antibody specificfor the other partner to detect anchored complexes. Again, dependingupon the order of addition of reactants to the liquid phase, testcompounds that inhibit complex or that disrupt preformed complexes canbe identified.

In an alternate embodiment of the invention, a homogeneous assay can beused. In this approach, a preformed complex of the endostatin geneprotein and the interactive binding partner is prepared in which eitherthe endostatin gene product or its binding partners is labeled, but thesignal generated by the label is quenched due to complex formation (see,e.g., U.S. Pat. No. 4,109,496 by Rubenstein which utilizes this approachfor immunoassays). The addition of a test substance that competes withand displaces one of the species from the preformed complex will resultin the generation of a signal above background. In this way, testsubstances that disrupt a endostatin gene protein/binding partnerinteraction can be identified.

In a particular embodiment, the endostatin gene product can be preparedfor immobilization using recombinant DNA techniques described above. Forexample, the endostatin coding region can be fused to aglutathione-S-transferase (GST) gene using a fusion vector, such aspGEX-5X-1, in such a manner that its binding activity is maintained inthe resulting fusion protein. The interactive binding partner can bepurified and used to raise a monoclonal antibody, using methodsroutinely practiced in the art and described above. This antibody can belabeled with the radioactive isotope 125I, for example, by methodsroutinely practiced in the art. In a heterogeneous assay, e.g., theGST-endostatin fusion protein can be anchored to glutathione-agarosebeads. The interactive binding partner can then be added in the presenceor absence of the test compound in a manner that allows interaction andbinding to occur. At the end of the reaction period, unbound materialcan be washed away, and the labeled monoclonal antibody can be added tothe system and allowed to bind to the complexed components. Theinteraction between the endostatin gene protein and the interactivebinding partner can be detected by measuring the amount of radioactivitythat remains associated with the glutathione-agarose beads. A successfulinhibition of the interaction by the test compound will result in adecrease in measured radioactivity.

Alternatively, the GST-endostatin gene fusion protein and theinteractive binding partner can be mixed together in liquid in theabsence of the solid glutathione-agarose beads. The test compound can beadded either during or after the species are allowed to interact. Thismixture can then be added to the glutathione-agarose beads and unboundmaterial is washed away. Again the extent of inhibition of theendostatin gene product/binding partner interaction can be detected byadding the labeled antibody and measuring the radioactivity associatedwith the beads.

In another embodiment of the invention, these same techniques can beemployed using peptide fragments that correspond to the binding domainsof the endostatin protein and/or the interactive or binding partner (incases where the binding partner is a protein), in place of one or bothof the full length proteins. Any number of methods routinely practicedin the art can be used to identify and isolate the binding sites. Thesemethods include, but are not limited to, mutagenesis of the geneencoding one of the proteins and screening for disruption of binding ina co-immunoprecipitation assay. Compensating mutations in the geneencoding the second species in the complex can then be selected.Sequence analysis of the genes encoding the respective proteins willreveal the mutations that correspond to the region of the proteininvolved in interactive binding. Alternatively, one protein can beanchored to a solid surface using methods described in this section,above, and allowed to interact with and bind to its labeled bindingpartner, which has been treated with a proteolytic enzyme, such astrypsin. After washing, a short, labeled peptide comprising the bindingdomain may remain associated with the solid material, which can beisolated and identified by amino acid sequencing. Also, once the genecoding for the segments can be engineered to express peptide fragmentsof the protein, which can then be tested for binding activity andpurified or synthesized.

For example, and not by way of limitation, an endostatin gene productcan be anchored to a solid material as described, above, in this sectionby making a GST-endostatin fusion protein and allowing it to bind toglutathione agarose beads. The interactive binding partner obtained canbe labeled with a radioactive isotope, such as 35S, and cleaved with aproteolytic enzyme such as trypsin. Cleavage products can then be addedto the anchored GST-endostatin fusion protein and allowed to bind. Afterwashing away unbound peptides, labeled bound material, representing thebinding partner binding domain, can be eluted, purified, and analyzedfor amino acid sequence by well-known methods. Peptides so identifiedcan be produced synthetically or fused to appropriate facilitativeproteins using recombinant DNA technology.

Compounds including, but not limited to, binding compounds identifiedvia assay techniques such as those described, above, can be tested forthe ability to ameliorate symptoms of an angiogenesis-related disorder,e.g., angiogenesis-dependent cancer, including, for example, solidtumors, blood born tumors such as leukemias, and tumor metastases;benign tumors, for example hemangiomas, acoustic neuromas,neurofibromas, trachomas, and pyogenic granulomas; rheumatoid arthritis;psoriasis; ocular angiogenic diseases, for example, diabeticretinopathy, retinopathy of prematurity, macular degeneration, cornealgraft rejection, neovascular glaucoma, retrolental fibroplasia,rubeosis; Osler-Webber Syndrome; myocardial angiogenesis; plaqueneovascularization; telangiectasia; hemophiliac joints; angiofibroma;wound granulation; corornary collaterals; cerebral collaterals;arteriovenous malformations; ischemic limb angiogenesis; diabeticneovascularization; macular degeneration; fractures; vasculogenesis;hematopoiesis; ovulation; menstruation; placentation; intestinaladhesions; atherosclerosis; scleroderma; hypertrophic scars, i.e.,keloids; cat scratch disease (Rochele minalia quintosa); and ulcers(Helobacter pylori). It should be noted that the assays described hereincan identify compounds that affect endostatin gene activity by eitheraffecting endostatin gene expression or by affecting the level ofendostatin gene product activity. For example, compounds may beidentified that are involved in another step in the pathway in which theendostatin gene and/or endostatin gene product is involved and, byaffecting this same pathway may modulate the effect of endostatin on thedevelopment of an angiogenesis-related disorder such as cancer. Suchcompounds can be used as part of a therapeutic method for the treatmentof the disorder.

Described below are cell-based and animal model-based assays for theidentification of compounds exhibiting such an ability to amelioratesymptoms of an angiogenesis-related disorder, e.g., cancer.

First, cell-based systems can be used to identify compounds that may actto ameliorate symptoms of an angiogenesis-related disorder, e.g.,cancer. Such cell systems can include, for example, recombinant ornon-recombinant cell, such as cell lines, that express the endostatingene.

In utilizing such cell systems, cells that express endostatin may beexposed to a compound suspected of exhibiting an ability to amelioratesymptoms of an angiogenesis-related disorder, e.g., cancer, at asufficient concentration and for a sufficient time to elicit such anamelioration of such symptoms in the exposed cells. After exposure, thecells can be assayed to measure alterations in the expression of theendostatin gene, e.g., by assaying cell lysates for endostatin mRNAtranscripts (e.g., by Northern analysis) or for endostatin gene productsexpressed by the cell; compounds that modulate expression of theendostatin gene are good candidates as therapeutics. Alternatively, thecells are examined to determine whether one or more cellular phenotypesassociated with an angiogenesis-related disorder, e.g., cancer, has beenaltered to resemble a more normal or unimpaired, unaffected phenotype,or a phenotype more likely to produce a lower incidence or severity ofdisorder symptoms.

In addition, animal-based systems or models for an angiogenesis-relateddisorder, e.g., cancer, may be used to identify compounds capable ofameliorating symptoms of the disorder. Such animal-based systems ormodels may include, for example, transgenic mice, e.g., mice that havebeen genetically engineered to express exogenous or endogenousendostatin sequences or, alternatively, to no longer express endogenousendostatin gene sequences (i.e., “knock-out” mice). Such animal modelsmay be used as test substrates for the identification of drugs,pharmaceuticals, therapies and interventions that may be effective intreating such disorders. For example, animal models may be exposed to acompound suspected of exhibiting an ability to ameliorate symptoms, at asufficient concentration and for a sufficient time to elicit such anamelioration of symptoms of an angiogenesis-related disorder, e.g.,cancer, in the exposed animals. The response of the animals to theexposure may be monitored by assessing the reversal of such symptoms.

With regard to intervention, any treatments that reverse any aspect ofsymptoms of an angiogenesis-related disorder, e.g., cancer, should beconsidered as candidates for human therapeutic intervention in such adisorder. Dosages of test agents may be determined by derivingdose-response curves, as discussed in, below.

A variety of methods can be employed for the diagnostic and prognosticevaluation of angiogenesis-related disorders, such as cancer, and forthe identification of subjects having a predisposition to suchdisorders.

Such methods may, for example, utilize reagents such as the endostatingene nucleotide sequences described above, and antibodies directedagainst endostatin gene products, including peptide fragments thereof,as described, above. Specifically, such reagents may be used, forexample, for:

-   -   (1) the detection of the presence of endostatin gene mutations,        or the detection of either over- or under-expression of        endostatin gene mRNA relative to the state of an        angiogenesis-related disorder, such as cancer;    -   (2) the detection of either an over- or an under-abundance of        endostatin gene product relative to the unaffected state; and    -   (3) the detection of an aberrant level of endostatin gene        product activity relative to the unaffected state.

Endostatin gene nucleotide sequences can, for example, be used todiagnose an angiogenesis-related disorder using, for example, thetechniques for endostatin mutation detection described above.

The methods described herein may be performed, for example, by utilizingpre-packaged diagnostic kits comprising at least one specific endostatingene nucleic acid or anti-endostatin gene antibody reagent describedherein, which may be conveniently used, e.g., in clinical settings, todiagnose subjects exhibiting abnormalities of an angiogenesis-relateddisorder, e.g., cancer.

For the detection of endostatin mutations, any nucleated cell can beused as a starting source for genomic nucleic acid. For the detection ofendostatin gene expression or endostatin gene products, any cell type ortissue in which the endostatin gene is expressed may be utilized.

Nucleic acid-based detection techniques are described, above. Peptidedetection techniques are described, above.

The methods described herein can furthermore be utilized as diagnosticor prognostic assays to identify subjects having or at risk ofdeveloping a disease or disorder associated with aberrant expression oractivity of a polypeptide of the invention. For example, the assaysdescribed herein, such as the preceding diagnostic assays or thefollowing assays, can be utilized to identify a subject having or atrisk of developing a disorder associated with aberrant expression oractivity of a polypeptide of the invention. Alternatively, theprognostic assays can be utilized to identify a subject having or atrisk for developing such a disease or disorder. Thus, the presentinvention provides a method in which a test sample is obtained from asubject and a polypeptide or nucleic acid (e.g., mRNA, genomic DNA) ofthe invention is detected, wherein the presence of the polypeptide ornucleic acid is diagnostic for a subject having or at risk of developinga disease or disorder associated with aberrant expression or activity ofthe polypeptide. As used herein, a “test sample” refers to a biologicalsample obtained from a subject of interest. For example, a test samplecan be a biological fluid (e.g., serum), cell sample, or tissue.

Furthermore, the prognostic assays described herein can be used todetermine whether a subject can be administered an agent (e.g., anagonist, antagonist, peptidomimetic, protein, peptide, nucleic acid,small molecule, or other drug candidate) to treat a disease or disorderassociated with aberrant expression or activity of a polypeptide of theinvention. For example, such methods can be used to determine whether asubject can be effectively treated with a specific agent or class ofagents (e.g., agents of a type which decrease activity of thepolypeptide). Thus, the present invention provides methods fordetermining whether a subject can be effectively treated with an agentfor a disorder associated with aberrant expression or activity of apolypeptide of the invention in which a test sample is obtained and thepolypeptide or nucleic acid encoding the polypeptide is detected (e.g.,wherein the: presence of the polypeptide or nucleic acid is diagnosticfor a subject that can be administered the agent to treat a disorderassociated with aberrant expression or activity of the polypeptide).

The methods of the invention also can be used to detect genetic lesionsor mutations in a gene of the invention, thereby determining if asubject with the lesioned gene is at risk for a disorder characterizedaberrant expression or activity of a polypeptide of the invention. Inpreferred embodiments, the methods include detecting, in a sample ofcells from the subject, the presence or absence of a genetic lesion ormutation characterized by at least one of an alteration affecting theintegrity of a gene encoding the polypeptide of the invention, or themis-expression of the gene encoding the polypeptide of the invention.For example, such genetic lesions or mutations can be detected byascertaining the existence of at least one of: 1) a deletion of one ormore nucleotides from the gene; 2) an addition of one or morenucleotides to the gene; 3) a substitution of one or more nucleotides ofthe gene; 4) a chromosomal rearrangement of the gene; 5) an alterationin the level of a messenger RNA transcript of the gene; 6) an aberrantmodification of the gene, such as of the methylation pattern of thegenomic DNA; 7) the presence of a non-wild type splicing pattern of amessenger RNA transcript of the gene; 8) a non-wild type level of a theprotein encoded by the gene; 9) an allelic loss of the gene; and 10) aninappropriate post-translational modification of the protein encoded bythe gene. As described herein, there are a large number of assaytechniques known in the art which can be used for detecting lesions in agene.

In certain embodiments, detection of the lesion involves the use of aprobe/primer in a polymerase chain reaction (PCR) (see, e.g., U.S. Pat.Nos. 4,683,195 and 4,683,202), such as anchor PCR or RACE PCR, or,alternatively, in a ligation chain reaction (LCR) (see, e.g., Landegranet al. (1988) Science 241:1077-1080; and Nakazawa et al. (1994) Proc.Natl. Acad. Sci. USA 91:360-364), the latter of which can beparticularly useful for detecting point mutations in a gene (see, e.g.,Abravaya et al. (1995) Nucleic Acids Res. 23:675-682). This method caninclude the steps of collecting a sample of cells from a subject,isolating nucleic acid (e.g., genomic, mRNA or both) from the cells ofthe sample, contacting the nucleic acid sample with one or more primerswhich specifically hybridize to the selected gene under conditions suchthat hybridization and amplification of the gene (if present) occurs,and detecting the presence or absence of an amplification product, ordetecting the size of the amplification product and comparing the lengthto a control sample. It is anticipated that PCR and/or LCR may bedesirable to use as a preliminary amplification step in conjunction withany of the techniques used for detecting mutations described herein.

Alternative amplification methods include: self sustained sequencereplication (Guatelli et al. (1990) Proc. Natl. Acad. Sci. USA87:1874-1878), transcriptional amplification system (Kwoh, et al. (1989)Proc. Natl. Acad. Sci. USA 86:1173-1177), Q-Beta Replicase (Lizardi etal. (1988) Bio/Technology 6:1197), or any other nucleic acidamplification method, followed by the detection of the amplifiedmolecules using techniques well known to those of skill in the art.These detection schemes are especially useful for the detection ofnucleic acid molecules if such molecules are present in very lownumbers.

In an alternative embodiment, mutations in a selected gene from a samplecell can be identified by alterations in restriction enzyme cleavagepatterns. For example, sample and control DNA is isolated, amplified(optionally), digested with one or more restriction endonucleases, andfragment length sizes are determined by gel electrophoresis andcompared. Differences in fragment length sizes between sample andcontrol DNA indicates mutations in the sample DNA. Moreover, the use ofsequence specific ribozymes (see, e.g., U.S. Pat. No. 5,498,531) can beused to score for the presence of specific mutations by development orloss of a ribozyme cleavage site.

In other embodiments, genetic mutations can be identified by hybridizinga sample and control nucleic acids, e.g., DNA or RNA, to high densityarrays containing hundreds or thousands of oligonucleotides probes(Cronin et al., 1996, Human Mutation 7:244-255; Kozal et al., 1996,Nature Medicine 2:753-759). For example, genetic mutations can beidentified in two-dimensional arrays containing light-generated DNAprobes as described in Cronin et al., supra. Briefly, a firsthybridization array of probes can be used to scan through long stretchesof DNA in a sample and control to identify base changes between thesequences by making linear arrays of sequential overlapping probes. Thisstep allows the identification of point mutations. This step is followedby a second hybridization array that allows the characterization ofspecific mutations by using smaller, specialized probe arrayscomplementary to all variants or mutations detected. Each mutation arrayis composed of parallel probe sets, one complementary to the wild-typegene and the other complementary to the mutant gene.

In yet another embodiment, any of a variety of sequencing reactionsknown in the art can be used to directly sequence the selected gene anddetect mutations by comparing the sequence of the sample nucleic acidswith the corresponding wild-type (control) sequence. (Examples ofsequencing reactions include those based on techniques developed byMaxim and Gilbert, 1977, Proc. Natl. Acad. Sci. USA 74:560 or Sanger,1977, Proc. Natl. Acad. Sci. USA 74:5463). It is also contemplated thatany of a variety of automated sequencing procedures can be utilized whenperforming the diagnostic assays (1995, Bio/Techniques 19:448),including sequencing by mass spectrometry (see, e.g., PCT PublicationNo. WO 94/16101; Cohen et al., 1996, Adv. Chromatogr. 36:127-162; andGriffin et al., 1993, Appl. Biochem. Biotechnol. 38:147-159).

Other methods for detecting mutations in a selected gene include methodsin which protection from cleavage agents is used to detect mismatchedbases in RNA/RNA or RNA/DNA heteroduplexes (Myers et al., 1985, Science230:1242). In general, the technique of mismatch cleavage entailsproviding heteroduplexes formed by hybridizing (labeled) RNA or DNAcontaining the wild-type sequence with potentially mutant RNA or DNAobtained from a tissue sample. The double-stranded duplexes are treatedwith an agent which cleaves single-stranded regions of the duplex suchas which will exist due to basepair mismatches between the control andsample strands. RNA/DNA duplexes can be treated with RNase to digestmismatched regions, and DNA/DNA hybrids can be treated with S1 nucleaseto digest mismatched regions. In other embodiments, either DNA/DNA orRNA/DNA duplexes can be treated with hydroxylamine or osmium tetroxideand with piperidine in order to digest mismatched regions. Afterdigestion of the mismatched regions, the resulting material is thenseparated by size on denaturing polyacrylamide gels to determine thesite of mutation. (See, e.g., Cotton et al., 1988, Proc. Natl. Acad.Sci. USA 85:4397; Saleeba et al., 1992, Methods Enzymol. 217:286-295.)In a preferred embodiment, the control DNA or RNA can be labeled fordetection.

In still another embodiment, the mismatch cleavage reaction employs oneor more proteins that recognize mismatched base pairs in double-strandedDNA (so called “DNA mismatch repair enzymes”) in defined systems fordetecting and mapping point mutations in cDNAs obtained from samples ofcells. For example, the mutY enzyme of E. coli cleaves A at G/Amismatches and the thymidine DNA glycosylase from HeLa cells cleaves Tat G/T mismatches (Hsu et al., 1994, Carcinogenesis 15:1657-1662).According to an exemplary embodiment, a probe based on a selectedsequence, e.g., a wild-type sequence, is hybridized to a cDNA or otherDNA product from a test cell(s). The duplex is treated with a DNAmismatch repair enzyme, and the cleavage products, if any, can bedetected from electrophoresis protocols or the like. (See, e.g., U.S.Pat. No. 5,459,039.)

In other embodiments, alterations in electrophoretic mobility will beused to identify mutations in genes. For example, SSCP may be used todetect differences in electrophoretic mobility between mutant and wildtype nucleic acids (Orita et al., 1989, Proc. Natl. Acad. Sci. USA86:2766; see also Cotton, 1993, Mutat. Res. 285:125-144; Hayashi, 1992,Genet. Anal. Tech. Appl. 9:73-79). Single-stranded DNA fragments ofsample and control nucleic acids will be denatured and allowed torenature. The secondary structure of single-stranded nucleic acidsvaries according to sequence, and the resulting alteration inelectrophoretic mobility enables the detection of even a single basechange. The DNA fragments may be labeled or detected with labeledprobes. The sensitivity of the assay may be enhanced by using RNA(rather than DNA), in which the secondary structure is more sensitive toa change in sequence. In a preferred embodiment, the subject methodutilizes heteroduplex analysis to separate double stranded heteroduplexmolecules on the basis of changes in electrophoretic mobility (Keen etal., 1991, Trends Genet. 7:5).

In yet another embodiment, the movement of mutant or wild-type fragmentsin polyacrylamide gels containing a gradient of denaturant is assayedusing denaturing gradient gel electrophoresis (DGGE) (Myers et al.,1985, Nature 313:495). When DGGE is used as the method of analysis, DNAwill be modified to insure that it does not completely denature, forexample by adding a GC clamp of approximately 40 bp of high-meltingGC-rich DNA by PCR. In a further embodiment, a temperature gradient isused in place of a denaturing gradient to identify differences in themobility of control and sample DNA (Rosenbaum and Reissner, 1987,Biophys. Chem. 265:12753).

Examples of other techniques for detecting point mutations include, butare not limited to, selective oligonucleotide hybridization, selectiveamplification, or selective primer extension. For example,oligonucleotide primers may be prepared in which the known mutation isplaced centrally and then hybridized to target DNA under conditionswhich permit hybridization only if a perfect match is found (Saiki etal., 1986, Nature 324:163; Saiki et al., 1989, Proc. Natl. Acad. Sci.USA 86:6230). Such allele specific oligonucleotides are hybridized toPCR amplified target DNA or a number of different mutations when theoligonucleotides are attached to the hybridizing membrane and hybridizedwith labeled target DNA.

Alternatively, allele specific amplification technology which depends onselective PCR amplification may be used in conjunction with the instantinvention. Oligonucleotides used as primers for specific amplificationmay carry the mutation of interest in the center of the molecule (sothat amplification depends on differential hybridization) (Gibbs et al.,1989, Nucleic Acids Res. 17:2437-2448) or at the extreme 3′ end of oneprimer where, under appropriate conditions, mismatch can prevent orreduce polymerase extension (Prossner, 1993, Tibtech 11:238). Inaddition, it may be desirable to introduce a novel restriction site inthe region of the mutation to create cleavage-based, detection(Gasparini et al., 1992, Mol. Cell Probes 6:1). It is anticipated thatin certain embodiments amplification may also be performed using Taqligase for amplification (Barany, 1991, Proc. Natl. Acad. Sci. USA88:189). In such cases, ligation will occur only if there is a perfectmatch at the 3′ end of the 5′ sequence making it possible to detect thepresence of a known mutation at a specific site by looking for thepresence or absence of amplification.

The methods described herein may be performed, for example, by utilizingpre-packaged diagnostic kits comprising at least one probe nucleic acidor antibody reagent described herein, which may be conveniently used,e.g., in clinical settings to diagnose subjects exhibiting symptoms orfamily history of a disease or illness involving a gene encoding apolypeptide of the invention. Furthermore, any cell type or tissue,preferably peripheral blood leukocytes, in which the polypeptide of theinvention is expressed may be utilized in the prognostic assaysdescribed herein.

The invention further provides kits that facilitate the use and/ordetection of endostatin genes and gene products described herein. Thekits described herein may be conveniently used, e.g., in clinicalsettings to diagnose subjects exhibiting symptoms or family history of adisease or illness involving a gene encoding a polypeptide of theinvention. Furthermore, any cell type or tissue in which the polypeptideof the invention is expressed may be utilized in the prognostic assaysdescribed herein.

In one embodiment, a diagnostic test kit for identifying cells ortissues which mis-express endostatin genes or gene products is provided.In this embodiment, a diagnostic kit is provided, with one or morecontainers comprising an oligonucleotide, e.g., a detectably labeledoligonucleotide, which hybridizes to a nucleic acid sequence encoding apolypeptide of the invention. In another embodiment, a kit is provided,with one or more containers comprising a pair of primers useful foramplifying a nucleic acid molecule encoding a polypeptide of theinvention. In various other embodiments, the kit can also comprise,e.g., a buffering agent, a preservative, or a protein stabilizing agent.The kit also can comprise components necessary for detecting thedetectable agent (e.g., an enzyme or a substrate). The kit also cancontain a control sample or a series of control samples which can beassayed and compared to the test sample. Each component of the kit isusually enclosed within an individual container and all of the variouscontainers are within a single package along with instructions forobserving whether the tested subject is suffering from, or is at risk ofdeveloping, a disorder associated with aberrant expression of thepolypeptide. Such a kit can be used, for example, to measure the levelsof a nucleic acid molecule encoding the protein in a sample of cellsfrom a subject, e.g., detecting mRNA levels or determining whether agene encoding the protein has been mutated or deleted.

In another embodiment, the invention provides kits for detecting thepresence of a polypeptide or nucleic acid of the invention in abiological sample (a test sample). Such kits can be used to determine ifa subject is suffering from or is at increased risk of developing adisorder associated with aberrant expression of a polypeptide of theinvention as discussed, for example, in sections above relating to usesof the sequences of the invention. In this embodiment, a kit isprovided, with one or more containers comprising: (1) a first antibody(e.g., attached to a solid support) which binds to a polypeptide of theinvention; and, optionally, (2) a second, different antibody which bindsto either the polypeptide or the first antibody and is conjugated to adetectable agent. Such kits can be used to determine if a subject issuffering from, or is at increased risk of, an angiogenesis-relateddisorder, such as cancer.

Described below are methods and compositions whereby anendostatin-mediated process can be modulated and/or whereby anangiogenesis-related disorder, e.g., cancer, may be treated.

For example, such methods can comprise administering compounds whichmodulate the expression of an endostatin gene and/or the synthesis oractivity of an endostatin gene product so that the process is modulatedor a symptom of the disorder is ameliorated.

Alternatively, in those instances whereby the angiogenesis-relateddisorder, e.g., cancer, results from endostatin gene mutations thatlower or abolish endostatin activity, respectively, such methods cancomprise supplying a mammal with a nucleic acid molecule encoding anunimpaired endostatin gene product such that an unimpaired endostatingene product is expressed and symptoms of the disorder are ameliorated.

In another embodiment of methods for the treatment of mammalianangiogenesis-related disorder, e.g., cancer, resulting from endostatingene mutations, such methods can comprise supplying a mammal with a cellcomprising a nucleic acid molecule that encodes an unimpaired endostatingene product such that the cell expresses the unimpaired endostatin geneproduct and symptoms of the disorder are ameliorated.

In cases in which a loss of normal endostatin gene product functionresults in the development of an angiogenesis-related disorderphenotype, e.g., cancer, an increase in endostatin gene product activitywould facilitate progress towards an asymptomatic state in individualorganisms exhibiting a deficient level of endostatin gene expressionand/or endostatin gene product activity. Methods for enhancing theexpression or synthesis of endostatin can include, for example, methodssuch as those described below.

Alternatively, symptoms of angiogenesis-related disorder phenotype,e.g., cancer, may be ameliorated by administering a compound thatdecreases the level of endostatin gene expression and/or endostatin geneproduct activity. Methods for inhibiting or reducing the level ofendostatin synthesis or expression can include, for example, methodssuch as those described below.

In one embodiment of treatment methods, the compounds administered donot comprise compounds, in particular drugs, reported to ameliorate orexacerbate the symptoms of an angiogenesis-related disorder. If suchtreatment methods do comprise such compounds, preferably such compoundsare utilized in a manner (e.g., different dosage, mode ofadministration, and/or co-administration with one or more additionalcompounds) that differs from the manner in which such compounds havebeen administered previously.

In another embodiment, symptoms of a disorder described herein, e.g.,cancer, may be ameliorated by endostatin protein therapy methods, e.g.,decreasing or increasing the level and/or activity of endostatin usingthe endostatin protein, fusion protein, and peptide sequences describedabove, or by the administration of proteins or protein fragments (e.g.,peptides) which interact with an endostatin gene or gene product andthereby inhibit or potentiate its activity.

Such protein therapy may include, for example, the administration of afunctional endostatin protein or fragments of an endostatin protein(e.g., peptides) which represent functional endostatin domains.

In one embodiment, endostatin fragments or peptides representing afunctional endostatin binding domain are administered to an individualorganism such that the peptides bind to an endostatin binding protein,e.g., an endostatin receptor. Such fragments or peptides may serve toinhibit endostatin activity in an individual organism by competing with,and thereby inhibiting, binding of endostatin to the binding protein,thereby ameliorating symptoms of a disorder described herein.Alternatively, such fragments or peptides may enhance endostatinactivity in an individual organism by mimicking the function ofendostatin in vivo, thereby ameliorating the symptoms of a disorderdescribed herein.

The proteins and peptides which may be used in the methods of theinvention include synthetic (e.g., recombinant or chemicallysynthesized) proteins and peptides, as well as naturally occurringproteins and peptides. The proteins and peptides may have both naturallyoccurring and non-naturally occurring amino acid residues (e.g., D-aminoacid residues) and/or one or more non-peptide bonds (e.g., imino, ester,hydrazide, semicarbazide, and azo bonds). The proteins or peptides mayalso contain additional chemical groups (i.e., functional groups)present at the amino and/or carboxy termini, such that, for example, thestability, bioavailability, and/or inhibitory activity of the peptide isenhanced. Exemplary functional groups include hydrophobic groups (e.g.carbobenzoxyl, dansyl, and t-butyloxycarbonyl, groups), an acetyl group,a 9-fluorenylmethoxy-carbonyl group, and macromolecular carrier groups(e.g., lipid-fatty acid conjugates, polyethylene glycol, orcarbohydrates) including peptide groups.

In another embodiment, symptoms of certain angiogenesis-relateddisorders, such as cancer, may be ameliorated by decreasing the level ofendostatin gene expression and/or endostatin gene product activity byusing endostatin gene sequences in conjunction with well-knownantisense, gene “knock-out,” ribozyme and/or triple helix methods todecrease the level of endostatin gene expression. Among the compoundsthat may exhibit the ability to modulate the activity, expression orsynthesis of the endostatin gene, including the ability to amelioratethe symptoms of an angiogenesis-related disorder, e.g., cancer, areantisense, ribozyme, and triple helix molecules. Such molecules may bedesigned to reduce or inhibit either unimpaired, or if appropriate,mutant target gene activity. Techniques for the production and use ofsuch molecules are well known to those of skill in the art.

Antisense RNA and DNA molecules act to directly block the translation ofmRNA by hybridizing to targeted mRNA and preventing protein translation.Antisense approaches involve the design of oligonucleotides that arecomplementary to a target gene mRNA. The antisense oligonucleotides willbind to the complementary target gene mRNA transcripts and preventtranslation. Absolute complementarity, although preferred, is notrequired.

A sequence “complementary” to a portion of an RNA, as referred toherein, means a sequence having sufficient complementarity to be able tohybridize with the RNA, forming a stable duplex; in the case ofdouble-stranded antisense nucleic acids, a single strand of the duplexDNA may thus be tested, or triplex formation may be assayed. The abilityto hybridize will depend on both the degree of complementarity and thelength of the antisense nucleic acid. Generally, the longer thehybridizing nucleic acid, the more base mismatches with an RNA it maycontain and still form a stable duplex (or triplex, as the case may be).One skilled in the art can ascertain a tolerable degree of mismatch byuse of standard procedures to determine the melting point of thehybridized complex.

In one embodiment, oligonucleotides complementary to non-coding regionsof the endostatin gene could be used in an antisense approach to inhibittranslation of endogenous endostatin mRNA. Antisense nucleic acidsshould be at least six nucleotides in length, and are preferablyoligonucleotides ranging from 6 to about 50 nucleotides in length. Inspecific aspects the oligonucleotide is at least 10 nucleotides, atleast 17 nucleotides, at least 25 nucleotides or at least 50nucleotides.

Regardless of the choice of target sequence, it is preferred that invitro studies are first performed to quantitate the ability of theantisense oligonucleotide to inhibit gene expression. It is preferredthat these studies utilize controls that distinguish between antisensegene inhibition and nonspecific biological effects of oligonucleotides.It is also preferred that these studies compare levels of the target RNAor protein with that of an internal control RNA or protein.Additionally, it is envisioned that results obtained using the antisenseoligonucleotide are compared with those obtained using a controloligonucleotide. It is preferred that the control oligonucleotide is ofapproximately the same length as the test oligonucleotide and that thenucleotide sequence of the oligonucleotide differs from the antisensesequence no more than is necessary to prevent specific hybridization tothe target sequence.

The oligonucleotides can be DNA or RNA or chimeric mixtures orderivatives or modified versions thereof, single-stranded ordouble-stranded. The oligonucleotide can be modified at the base moiety,sugar moiety, or phosphate backbone, for example, to improve stabilityof the molecule, hybridization, etc. The oligonucleotide may includeother appended groups such as peptides (e.g., for targeting host cellreceptors in vivo), or agents facilitating transport across the cellmembrane (see, e.g., Letsinger, et al., 1989, Proc. Natl. Acad. Sci.U.S.A. 86, 6553-6556; Lemaitre, et al., 1987, Proc. Natl. Acad. Sci. 84,648-652; PCT Publication No. WO88/09810, published Dec. 15, 1988) or theblood-brain barrier (see, e.g., PCT Publication No. WO89/10134,published Apr. 25, 1988), hybridization-triggered cleavage agents (see,e.g., Krol et al., 1988, BioTechniques 6, 958-976) or intercalatingagents (see, e.g., Zon, 1988, Pharm. Res. 5, 539-549). To this end, theoligonucleotide may be conjugated to another molecule, e.g., a peptide,hybridization triggered cross-linking agent, transport agent,hybridization-triggered cleavage agent, etc.

The antisense oligonucleotide may comprise at least one modified basemoiety which is selected from the group including but not limited to5-fluorouracil, 5-bromouracil, 5-chlorouracil, 5-iodouracil,hypoxanthine, xanthine, 4-acetylcytqsine, 5-(carboxyhydroxylmethyl)uracil, 5-carboxymethylaminomethyl-2-thiouridine,5-carboxymethylaminomethyluracil, dihydrouracil,beta-D-galactosylqueosine, inosine, N6-isopentenyladenine,1-methylguanine, 1-methylinosine, 2,2-dimethylguanine, 2-methyladenine,2-methylguanine, 3-methylcytosine, 5-methylcytosine, N6-adenine,7-methylguanine, 5-methylaminomethyluracil,5-methoxyaminomethyl-2-thiouracil, beta-D-mannosylqueosine,5-methoxycarboxymethyluracil, 5-methoxyuracil,2-methylthio-N6-isopentenyladenine, uracil-5-oxyacetic acid (v),wybutoxosine, pseudouracil, queosine, 2-thiocytosine,5-methyl-2-thiouracil, 2-thiouracil, 4-thiouracil, 5-methyluracil,uracil-5-oxyacetic acid methylester, uracil-5-oxyacetic acid (v),5-methyl-2-thiouracil, 3-(3-amino-3-N-2-carboxypropyl) uracil, (acp3)w,and 2,6-diaminopurine.

The antisense oligonucleotide may also comprise at least one modifiedsugar moiety selected from the group including but not limited toarabinose, 2-fluoroarabinose, xylulose, and hexose.

In yet another embodiment, the antisense oligonucleotide comprises atleast one modified phosphate backbone selected from the group consistingof a phosphorothioate, a phosphorodithioate, a phosphoramidothioate, aphosphoramidate, a phosphordiamidate, a methylphosphonate, an alkylphosphotriester, and a formacetal or analog thereof.

In yet another embodiment, the antisense oligonucleotide is anα-anomeric oligonucleotide. An α-anomeric oligonucleotide forms specificdouble-stranded hybrids with complementary RNA in which, contrary to theusual β-units, the strands run parallel to each other (Gautier, et al.,1987, Nucl. Acids Res. 15, 6625-6641). The oligonucleotide is a2′-O-methylribonucleotide (Inoue, et al., 1987, Nucl. Acids Res. 15,6131-6148), or a chimeric RNA-DNA analogue (Inoue, et al., 1987, FEBSLett. 215, 327-330).

Oligonucleotides of the invention may be synthesized by standard methodsknown in the art, e.g. by use of an automated DNA synthesizer (such asare commercially available from Biosearch, Applied Biosystems, etc.). Asexamples, phosphorothioate oligonucleotides may be synthesized by themethod of Stein, et al. (1988, Nucl. Acids Res. 16, 3209),methylphosphonate oligonucleotides can be prepared by use of controlledpore glass polymer supports (Sarin, et al., 1988, Proc. Natl. Acad. Sci.U.S.A. 85, 7448-7451), etc.

While antisense nucleotides complementary to the target gene codingregion sequence could be used, those complementary to the transcribed,untranslated region are most preferred.

Antisense molecules should be delivered to cells that express the targetgene in vivo. A number of methods have been developed for deliveringantisense DNA or RNA to cells; e.g., antisense molecules can be injecteddirectly into the tissue site, or modified antisense molecules, designedto target the desired cells (e.g., antisense linked to peptides orantibodies that specifically bind receptors or antigens expressed on thetarget cell surface) can be administered systemically.

However, it is often difficult to achieve intracellular concentrationsof the antisense sufficient to suppress translation of endogenous mRNAs.Therefore a preferred approach utilizes a recombinant DNA-construct inwhich the antisense oligonucleotide is placed under the control of astrong pol III or pol II promoter. The use of such a construct totransfect target cells in the subject will result in the transcriptionof sufficient amounts of single stranded RNAs that will formcomplementary base pairs with the endogenous target gene transcripts andthereby prevent translation of the target gene mRNA. For example, avector can be introduced e.g., such that it is taken up by a cell anddirects the transcription of an antisense RNA. Such a vector can remainepisomal or become chromosomally integrated, as long as it can betranscribed to produce the desired antisense RNA. Such vectors can beconstructed by recombinant DNA technology methods standard in the art.Vectors can be plasmid, viral, or others known in the art, used forreplication and expression in mammalian cells. Expression of thesequence encoding the antisense RNA can be by any promoter known in theart to act in mammalian, preferably human cells. Such promoters can beinducible or constitutive. Such promoters include but are not limitedto: the SV40 early promoter region I(Benoist and Chambon, 1981, Nature290, 304-310), the promoter contained in the 3′ long terminal repeat ofRous sarcoma virus (Yamamoto, et al., 1980, Cell 22, 787-797), theherpes thymidine kinase promoter (Wagner, et al., 1981, Proc. Natl.Acad. Sci. U.S.A. 78, 1441-1445), the regulatory sequences of themetallothionein gene (Brinster, et al., 1982, Nature 296, 3942), etc.Any type of plasmid, cosmid, YAC or viral vector can be used to preparethe recombinant DNA construct which can be introduced directly into thetissue site. Alternatively, viral vectors can be used that selectivelyinfect the desired tissue, in which case administration may beaccomplished by another route (e.g., systemically).

Ribozyme molecules designed to catalytically cleave target gene mRNAtranscripts can also be used to prevent translation of target gene mRNAand, therefore, expression of target gene product. (See, e.g., PCTInternational Publication WO90/11364, published Oct. 4, 1990; Sarver, etal., 1990, Science 247, 1222-1225).

Ribozymes are enzymatic RNA molecules capable of catalyzing the specificcleavage of RNA. (For a review, see Rossi, 1994, Current Biology 4,469-471). The mechanism of ribozyme action involves sequence specifichybridization of the ribozyme molecule to complementary target RNA,followed by an endonucleolytic cleavage event. The composition ofribozyme molecules must include one or more sequences complementary tothe target gene mRNA, and must include the well known catalytic sequenceresponsible for mRNA cleavage. For this sequence, see, e.g., U.S. Pat.No. 5,093,246, which is incorporated herein by reference in itsentirety.

While ribozymes that cleave mRNA at site specific recognition sequencescan be used to destroy target gene mRNAs, the use of hammerheadribozymes is preferred. Hammerhead ribozymes cleave mRNAs at locationsdictated by flanking regions that form complementary base pairs with thetarget mRNA. The sole requirement is that the target mRNA have thefollowing sequence of two bases: 5′-UG-3′. The construction andproduction of hammerhead ribozymes is well known in the art and isdescribed more fully in Myers, 1995, Molecular Biology andBiotechnology: A Comprehensive Desk Reference, VCH Publishers, New York,(see especially FIG. 4, page 833) and in Haseloff and Gerlach, 1988,Nature, 334, 585-591, which is incorporated herein by reference in itsentirety.

Preferably the ribozyme is engineered so that the cleavage recognitionsite is located near the 5′ end of the target gene mRNA, i.e., toincrease efficiency and minimize the intracellular accumulation ofnon-functional mRNA transcripts.

The ribozymes of the present invention also include RNAendoribonucleases (hereinafter “Cech-type ribozymes”) such as the onethat occurs naturally in Tetrahymena thermophila (known as the IVS, orL-19 IVS RNA) and that has been extensively described by Thomas Cech andcollaborators (Zaug, et al., 1984, Science, 224, 574-578; Zaug and Cech,1986, Science, 231, 470-475; Zaug, et al., 1986, Nature, 324, 429-433;published International patent application No. WO 88/04300 by UniversityPatents Inc.; Been and Cech, 1986, Cell, 47, 207-216). The Cech-typeribozymes have an eight base pair active site which hybridizes to atarget RNA sequence whereafter cleavage of the target RNA takes place.The invention encompasses those Cech-type ribozymes which target eightbase-pair active site sequences that are present in the target gene.

As in the antisense approach, the ribozymes can be composed of modifiedoligonucleotides, (e.g., for improved stability, targeting, etc.) andshould be delivered to cells that express the target gene in vivo. Apreferred method of delivery involves using a DNA construct “encoding”the ribozyme under the control of a strong constitutive pol III or polII promoter, so that transfected cells will produce sufficientquantities of the ribozyme to destroy endogenous target gene messagesand inhibit translation. Because ribozymes, unlike antisense molecules,are catalytic, a lower intracellular concentration is required forefficiency.

Endogenous target gene expression can also be reduced by inactivating or“knocking out” the target gene or its promoter using targeted homologousrecombination (e.g., see Smithies, et al., 1985, Nature 317, 230-234;Thomas and Capecchi, 1987, Cell 51, 503-512; Thompson, et al., 1989,Cell 5, 313-321; each of which is incorporated by reference herein inits entirety). For example, a mutant, non-functional target gene (or acompletely unrelated DNA sequence) flanked by DNA homologous to theendogenous target gene (either the coding regions or regulatory regionsof the target gene) can be used, with or without a selectable markerand/or a negative selectable marker, to transfect cells that express thetarget gene in vivo. Insertion of the DNA construct, via targetedhomologous recombination, results in inactivation of the target gene.Such approaches are particularly suited in the agricultural field wheremodifications to ES (embryonic stem) cells can be used to generateanimal offspring with an inactive target gene (e.g., see Thomas andCapecchi, 1987 and Thompson, 1989, supra). However this approach can beadapted for use in humans provided the recombinant DNA constructs aredirectly administered or targeted to the required site in vivo usingappropriate viral vectors.

Alternatively, endogenous target gene expression can be reduced bytargeting deoxyribonucleotide sequences complementary to the regulatoryregion of the target gene (i.e., the target gene promoter and/orenhancers) to form triple helical structures that prevent transcriptionof the target gene in target cells in the body. (See generally, Helene,1991, Anticancer Drug Des., 6(6), 569-584; Helene, et al., 1992, Ann.N.Y. Acad. Sci., 660, 27-36; and Maher, 1992, Bioassays 14(12),807-815).

Nucleic acid molecules to be used in triplex helix formation for theinhibition of transcription should be single stranded and composed ofdeoxynucleotides. The base composition of these oligonucleotides must bedesigned to promote triple helix formation via Hoogsteen base pairingrules, which generally require sizeable stretches of either purines orpyrimidines to be present on one strand of a duplex. Nucleotidesequences may be pyrimidine-based, which will result in TAT and CGCtriplets across the three associated strands of the resulting triplehelix. The pyrimidine-rich molecules provide base complementarity to apurine-rich region of a single strand of the duplex in a parallelorientation to that strand. In addition, nucleic acid molecules may bechosen that are purine-rich, for example, contain a stretch of Gresidues. These molecules will form a triple helix with a DNA duplexthat is rich in GC pairs, in which the majority of the purine residuesare located on a single strand of the targeted duplex, resulting in GGCtriplets across the three strands in the triplex.

Alternatively, the potential sequences that can be targeted for triplehelix formation may be increased by creating a so called “switchback”nucleic acid molecule. Switchback molecules are synthesized in analternating 5′-3′, 3′-5′ manner, such that they base pair with first onestrand of a duplex and then the other, eliminating the necessity for asizeable stretch of either purines or pyrimidines to be present on onestrand of a duplex.

In instances wherein the antisense, ribozyme, and/or triple helixmolecules described herein are utilized to inhibit mutant geneexpression, it is possible that the technique may so efficiently reduceor inhibit the transcription (triple helix) and/or translation(antisense, ribozyme) of mRNA produced by normal target gene allelesthat the possibility may arise wherein the concentration of normaltarget gene product present may be lower than is necessary for a normalphenotype. In such cases, to ensure that substantially normal levels oftarget gene activity are maintained, therefore, nucleic acid moleculesthat encode and express target gene polypeptides exhibiting normaltarget gene activity may, be introduced into cells via gene therapymethods such as those described, below, that do not contain sequencessusceptible to whatever antisense, ribozyme, or triple helix treatmentsare being utilized. Alternatively, in instances whereby the target geneencodes an extracellular protein, it may be preferable to co-administernormal target gene protein in order to maintain the requisite level oftarget gene activity.

Anti-sense RNA and DNA, ribozyme, and triple helix molecules of theinvention may be prepared by any method known in the art for thesynthesis of DNA and RNA molecules, as discussed above. These includetechniques for chemically synthesizing oligodeoxyri-bonucleotides andoligoribonucleotides well known in the art such as for example solidphase phosphoramidite chemical synthesis. Alternatively, RNA moleculesmay be generated by in vitro and in vivo transcription of DNA sequencesencoding the antisense RNA molecule. Such DNA sequences may beincorporated into a wide variety of vectors that incorporate suitableRNA polymerase promoters such as the T7 or SP6 polymerase promoters.Alternatively, antisense cDNA constructs that synthesize antisense RNAconstitutively or inducibly, depending on the promoter used, can beintroduced stably into cell lines.

With respect to an increase in the level of normal endostatin geneexpression and/or endostatin gene product activity, endostatin genenucleic acid sequences, described above, for example, can be utilizedfor the treatment of an angiogenesis-related disorder, e.g., cancer.Such treatment can be administered, for example, in the form of genereplacement therapy. Specifically, one or more copies of a normalendostatin gene or a portion of the endostatin gene that directs theproduction of a endostatin gene product exhibiting normal endostatingene function, may be inserted into the appropriate cells within asubject, using vectors that include, but are not limited to, adenovirus,adeno-associated virus, herpesvirus and retrovirus vectors, in additionto other particles that introduce DNA into cells, such as liposomes.

Because endostatin genes can be expressed in the brain, such genereplacement therapy techniques should be capable delivering endostatingene sequences to these cell types within subjects. Thus, in oneembodiment, techniques that are well known to those of skill in the art(see, e.g., PCT Publication No. WO89/10134, published Apr. 25, 1988) canbe used to enable endostatin gene sequences to cross the blood-brainbarrier readily and to deliver the sequences to cells in the brain. Withrespect to delivery that is capable of crossing the blood-brain barrier,viral vectors such as, for example, those described above, arepreferable.

In another embodiment, techniques for delivery involve directadministration of such endostatin gene sequences to the site of thecells in which the endostatin gene, sequences are to be expressed.

Additional methods that may be utilized to increase the overall level ofendostatin gene expression and/or endostatin gene product activityinclude the introduction of appropriate endostatin-expressing cells,preferably autologous cells, into a subject at positions and in numbersthat are sufficient to ameliorate the symptoms of anangiogenesis-related disorder, e.g., cancer. Such cells may be eitherrecombinant or non-recombinant.

Among the cells that can be administered to increase the overall levelof endostatin gene expression in a subject are normal cells, preferablyliver cells, that express the endostatin gene.

Alternatively, cells, preferably autologous cells, can be engineered toexpress endostatin gene sequences, and may then be introduced into asubject in positions appropriate for the amelioration of the symptoms ofan angiogenesis-related disorder, e.g., cancer. Alternately, cells thatexpress an unimpaired endostatin gene and that are from an MHC matchedindividual organism can be utilized, and may include, for example, livercells. The expression of the endostatin gene sequences is controlled bythe appropriate gene regulatory sequences to allow such expression inthe necessary cell types. Such gene regulatory sequences are well knownto the skilled artisan. Such cell-based gene therapy techniques are wellknown to those skilled in the art, see, e.g., Anderson, U.S. Pat. No.5,399,349.

When the cells to be administered are non-autologous cells, they can beadministered using well known techniques that prevent a host immuneresponse against the introduced cells from developing. For example, thecells may be introduced in an encapsulated form which, while allowingfor an exchange of components with the immediate extracellularenvironment, does not allow the introduced cells to be recognized by thehost immune system.

Additionally, compounds, such as those identified via techniques such asthose described above, that are capable of modulating endostatin geneproduct activity can be administered using standard techniques that arewell known to those of skill in the art. In instances in which thecompounds to be administered are to involve an interaction with braincells, the administration techniques should include well known ones thatallow for a crossing of the blood-brain barrier.

Agents, or modulators, which have a stimulatory or inhibitory effect onactivity or expression of a polypeptide of the invention as identifiedby a screening assay described herein can be administered to individualorganisms to treat (prophylactically or therapeutically) disordersassociated with aberrant activity of the polypeptide. In conjunctionwith such treatment, the pharmacogenomics (i.e., the study of therelationship between an individual's genotype and that individual'sresponse to a foreign compound or drug) of the individual organism maybe considered. Differences in metabolism of therapeutics can lead tosevere toxicity or therapeutic failure by altering the relation betweendose and blood concentration of the pharmacologically active drug. Thus,the pharmacogenomics of the individual organism permits the selection ofeffective agents (e.g., drugs) for prophylactic or therapeutictreatments based on a consideration of the individual organism'sgenotype. Such pharmacogenomics can further be used to determineappropriate dosages and therapeutic regimens. Accordingly, the activityof a polypeptide of the invention, expression of a nucleic acid of theinvention, or mutation content of a gene of the invention in anindividual organism can be determined to thereby select appropriateagent(s) for therapeutic or prophylactic treatment of the individualorganism.

Pharmacogenomics deals with clinically significant hereditary variationsin the response to drugs due to altered drug disposition and abnormalaction in affected individual organisms. See, e.g., Linder (1997) Clin.Chem. 43(2):254-266. In general, two types of pharmacogenetic conditionscan be differentiated. Genetic conditions transmitted as a single factoraltering the way drugs act on the body are referred to as “altered drugaction.” Genetic conditions transmitted as single factors altering theway the body acts on drugs are referred to as “altered drug metabolism”.These pharmacogenetic conditions can occur either as rare defects or aspolymorphisms. For example, glucose-6-phosphate dehydrogenase deficiency(G6PD) is a common inherited enzymopathy in which the main clinicalcomplication is haemolysis after ingestion of oxidant drugs(anti-malarials, sulfonamides, analgesics, nitrofurans) and consumptionof fava beans.

As an illustrative embodiment, the activity of drug metabolizing enzymesis a major determinant of both the intensity and duration of drugaction. The discovery of genetic polymorphisms of drug metabolizingenzymes (e.g., N-acetyltransferase 2 (NAT 2) and cytochrome P450 enzymesCYP2D6 and CYP2C19) has provided an explanation as to why some subjectsdo not obtain the expected drug effects or show exaggerated drugresponse and serious toxicity after taking the standard and safe dose ofa drug. These polymorphisms are expressed in two phenotypes in thepopulation, the extensive metabolizer (EM) and poor metabolizer (PM).The prevalence of PM is different among different populations. Forexample, the gene coding for CYP2D6 is highly polymorphic and severalmutations have been identified in PM, which all lead to the absence offunctional CYP2D6. Poor metabolizers of CYP2D6 and CYP2C19 quitefrequently experience exaggerated drug response and side effects whenthey receive standard doses. If a metabolite is the active therapeuticmoiety, a PM will show no therapeutic response, as demonstrated for theanalgesic effect of codeine mediated by its CYP2D6-formed metabolitemorphine. The other extreme are the so called ultra-rapid metabolizerswho do not respond to standard doses. Recently, the molecular basis ofultra-rapid metabolism has been identified to be due to CYP2D6 geneamplification.

Thus, the activity of a polypeptide of the invention, expression of anucleic acid encoding the polypeptide, or mutation content of a geneencoding the polypeptide in an individual organism can be determined tothereby select appropriate agent(s) for therapeutic or prophylactictreatment of the individual organism. In addition, pharmacogeneticstudies can be used to apply genotyping of polymorphic alleles encodingdrug-metabolizing enzymes to the identification of an individualorganism's drug responsiveness phenotype. This knowledge, when appliedto dosing or drug selection, can avoid adverse reactions or therapeuticfailure and thus enhance therapeutic or prophylactic efficiency whentreating a subject with a modulator of activity or expression of thepolypeptide, such as a modulator identified by one of the exemplaryscreening assays described herein.

Monitoring the influence of agents (e.g., drugs, compounds) on theexpression or activity of a polypeptide of the invention (e.g., theability to modulate aberrant cell proliferation chemotaxis, and/ordifferentiation) can be applied not only in basic drug screening, butalso in clinical trials. For example, the effectiveness of an agent, asdetermined by a screening assay as described herein, to increase geneexpression, protein levels or protein activity, can be monitored inclinical trials of subjects exhibiting decreased gene expression,protein levels, or protein activity. Alternatively, the effectiveness ofan agent, as determined by a screening assay, to decrease geneexpression, protein levels or protein activity, can be monitored inclinical trials of subjects exhibiting increased gene expression,protein levels, or protein activity. In such clinical trials, expressionor activity of a polypeptide of the invention and preferably, that ofother polypeptide that have been implicated in for example, a cellularproliferation disorder, can be used as a marker of the immuneresponsiveness of a particular cell.

For example, and not by way of limitation, genes, including those of theinvention, that are modulated in cells by treatment with an agent (e.g.,compound, drug or small molecule) which modulates activity or expressionof a polypeptide of the invention (e.g., as identified in a screeningassay described herein) can be identified. Thus, to study the effect ofagents on cellular proliferation disorders, for example, in a clinicaltrial, cells can be isolated and RNA prepared and analyzed for thelevels of expression of a gene of the invention and other genesimplicated in the disorder. The levels of gene expression (i.e., a geneexpression pattern) can be quantified by Northern blot analysis orRT-PCR, as described herein; or alternatively by measuring the amount ofprotein produced, by one of the methods as described herein, or bymeasuring the levels of activity of a gene of the invention or othergenes. In this way, the gene expression pattern can serve as a marker,indicative of the physiological response of the cells to the agent.Accordingly, this response state may be determined before, and atvarious points during, treatment of the individual organism with theagent.

In a preferred embodiment, the present invention provides a method formonitoring the effectiveness of treatment of a subject with an agent(e.g., an agonist, antagonist, peptidomimetic, protein, peptide, nucleicacid, small molecule, or other drug candidate identified by thescreening assays described herein) comprising the steps of (i) obtaininga pre-administration sample from a subject prior to administration ofthe agent; (ii) detecting the level of the polypeptide or nucleic acidof the invention in the preadministration sample; (iii) obtaining one ormore post-administration samples from the subject; (iv) detecting thelevel the of the polypeptide or nucleic acid of the invention in thepost-administration samples; (v) comparing the level of the polypeptideor nucleic acid of the invention in the pre-administration sample withthe level of the polypeptide or nucleic acid of the invention in thepost-administration sample or samples; and (vi) altering theadministration of the agent to the subject accordingly. For example,increased administration of the agent may be desirable to increase theexpression or activity of the polypeptide to higher levels thandetected, i.e., to increase the effectiveness of the agent.Alternatively, decreased administration of the agent may be desirable todecrease expression or activity of the polypeptide to lower levels thandetected, i.e., to decrease the effectiveness of the agent.

The compounds of this invention can be formulated and administered toinhibit a variety of angiogenesis-related disorders by any means thatproduces contact of the active ingredient with the agents site of actionin the body of a mammal. They can be administered by any conventionalmeans available for use in conjunction with pharmaceuticals, either asindividual therapeutic active ingredients or in a combination oftherapeutic active ingredients. They can be administered alone, but aregenerally administered with a pharmaceutical carrier selected on thebasis of the chosen route of administration and standard pharmaceuticalpractice.

The dosage administered will be a therapeutically effective amount ofthe compound sufficient to result in amelioration of symptoms of theangiogenesis-related disorder and will, of course, vary depending uponknown factors such as the pharmacodynamic characteristics of theparticular active ingredient and its mode and route of administration;age, sex, health and weight of the recipient; nature and extent ofsymptoms; kind of concurrent treatment, frequency of treatment and theeffect desired.

Toxicity and therapeutic efficacy of such compounds can be determined bystandard pharmaceutical procedures in cell cultures or experimentalanimals, e.g., for determining the LD50 (the dose lethal to 50% of thepopulation) and the ED50 (the dose therapeutically effective in 50% ofthe population). The dose ratio between toxic and therapeutic effects isthe therapeutic index and it can be expressed as the ratio LD50/ED50.Compounds which exhibit large therapeutic indices are preferred. Whilecompounds that exhibit toxic side effects may be used, care should betaken to design a delivery system that targets such compounds to thesite of affected tissue in order to minimize potential damage touninfected cells and, thereby, reduce side effects.

The data obtained from the cell culture assays and animal studies can beused in formulating a range of dosage for use in humans. The dosage ofsuch compounds lies preferably within a range of circulatingconcentrations that include the ED50 with little or no toxicity. Thedosage may vary within this range depending upon the dosage formemployed and the route of administration utilized. For any compound usedin the method of the invention, the therapeutically effective dose canbe estimated initially from cell culture assays. A dose may beformulated in animal models to achieve a circulating plasmaconcentration range that includes the IC50 (i.e., the concentration ofthe test compound which achieves a half-maximal inhibition of symptoms)as determined in cell culture. Such information can be used to moreaccurately determine useful doses in humans. Levels in plasma may bemeasured, for example, by high performance liquid chromatography.

Specific dosages may also be utilized for antibodies. Typically, thepreferred dosage is 0.1 mg/kg to 100 mg/kg of body weight (generally 10mg/kg to 20 mg/kg), and if the antibody is to act in the brain, a dosageof 50 mg/kg to 100 mg/kg is usually appropriate. If the antibody ispartially human or fully human, it generally will have a longerhalf-life within the human body than other antibodies. Accordingly,lower dosages of partially human and fully human antibodies is oftenpossible. Additional modifications may be used to further stabilizeantibodies. For example, lipidation can be used to stabilize antibodiesand to enhance uptake and tissue penetration (e.g., into the brain). Amethod for lipidation of antibodies is described by Cruikshank et al.((1997) J. Acquired Immune Deficiency Syndromes and Human Retrovirology14:193).

A therapeutically effective amount of protein or polypeptide (i.e., aneffective dosage) ranges from about 0.001 to 30 mg/kg body weight,preferably about 0.01 to 25 mg/kg body weight, more preferably about 0.1to 20 mg/kg body weight, and even more preferably about 1 to 10 mg/kg, 2to 9 mg/kg, 3 to 8 mg/kg, 4 to 7 mg/kg, or 5 to 6 mg/kg body weight.

Moreover, treatment of a subject with a therapeutically effective amountof a protein, polypeptide or antibody can include a single treatment or,preferably, can include a series of treatments. In a preferred example,a subject is treated with antibody, protein, or polypeptide in the rangeof between about 0.1 to 20 mg/kg body weight, one time per week forbetween about 1 to 10 weeks, preferably between 2 to 8 weeks, morepreferably between about 3 to 7 weeks, and even more preferably forabout 4, 5 or 6 weeks.

The present invention further encompasses agents which modulateexpression or activity. An agent may, for example, be a small molecule.For example, such small molecules include, but are not limited to,peptides, peptidomimetics, amino acids, amino acid analogs,polynucleotides, polynucleotide analogs, nucleotides, nucleotideanalogs, organic or inorganic compounds (i.e,. including heteroorganicand organometallic compounds) having a molecular weight less than about10,000 grams per mole, organic or inorganic compounds having a molecularweight less than about 5,000 grams per mole, organic or inorganiccompounds having a molecular weight less than about 1,000 grams permole, organic or inorganic compounds having a molecular weight less thanabout 500 grams per mole, and salts, esters, and other pharmaceuticallyacceptable forms of such compounds.

It is understood that appropriate doses of small molecule agents dependsupon a number of factors known to those or ordinary skill in the art,e.g., a physician. The dose(s) of the small molecule will vary, forexample, depending upon the identity, size, and condition of the subjector sample being treated, further depending upon the route by which thecomposition is to be administered, if applicable, and the effect whichthe practitioner desires the small molecule to have upon the nucleicacid or polypeptide of the invention. Exemplary doses include milligramor microgram amounts of the small molecule per kilogram of subject orsample weight (e.g., about 1 microgram per kilogram to about 500milligrams per kilogram, about 100 micrograms per kilogram to about 5milligrams per kilogram, or about 1 microgram per kilogram to about 50micrograms per kilogram.

Pharmaceutical compositions for use in accordance with the presentinvention may be formulated in conventional manner using one or morephysiologically acceptable carriers or excipients.

Thus, the compounds and their physiologically acceptable salts andsolvates may be formulated for administration by inhalation orinsulation (either through the mouth or the nose) or oral, buccal,parenteral or rectal administration.

For oral administration, the pharmaceutical compositions may take theform of, for example, tablets or capsules prepared by conventional meanswith pharmaceutically acceptable excipients such as binding agents(e.g., pregelatinised maize starch, polyvinylpyrrolidone orhydroxypropyl methylcellulose); fillers (e.g., lactose, microcrystallinecellulose or calcium hydrogen phosphate); lubricants (e.g., magnesiumstearate, talc or silica); disintegrants (e.g., potato starch or sodiumstarch glycolate); or wetting agents (e.g., sodium lauryl sulphate). Thetablets may be coated by methods well known in the art. Liquidpreparations for oral administration may take the form of, for example,solutions, syrups or suspensions, or they may be presented as a dryproduct for constitution with water or other suitable vehicle beforeuse. Such liquid preparations may be prepared by conventional means withpharmaceutically acceptable additives such as suspending agents (e.g.,sorbitol syrup, cellulose derivatives or hydrogenated edible fats);emulsifying agents (e.g., lecithin or acacia); non-aqueous vehicles(e.g., almond oil, oily esters, ethyl alcohol or fractionated vegetableoils); and preservatives (e.g., methyl or propyl-p-hydroxybenzoates orsorbic acid). The preparations may also contain buffer salts, flavoring,coloring and sweetening agents as appropriate.

Preparations for oral administration may be suitably formulated to givecontrolled release of the active compound.

For buccal administration the compositions may take the form of tabletsor lozenges formulated in conventional manner.

For administration by inhalation, the compounds for use according to thepresent invention are conveniently delivered in the form of an aerosolspray presentation from pressurized packs or a nebulizer, with the useof a suitable propellant, egg., dichlorodifluoromethane,trichlorofluoromethane, dichlorotetrafluoroethane, carbon dioxide orother suitable gas. In the case of a pressurized aerosol the dosage unitmay be determined by providing a valve to deliver a metered amount.Capsules and cartridges of e.g. gelatin for use in an inhaler orinsulator may be formulated containing a powder mix of the compound anda suitable powder base such as lactose or starch.

The compounds may be formulated for parenteral administration byinjection, e.g., by bolus injection or continuous infusion. Formulationsfor injection may be presented in unit dosage form, e.g., in ampoules orin multi-dose containers, with an added preservative. The compositionsmay take such forms as suspensions, solutions or emulsions in oily oraqueous vehicles, and may contain formulatory agents such as suspending,stabilizing and/or dispersing agents. Alternatively, the activeingredient may be in powder form for constitution with a suitablevehicle, e.g., sterile pyrogen-free water, before use. In general,water, a suitable oil, saline, aqueous dextrose (glucose), and relatedsugar solutions and glycols such as propylene glycol or polyethyleneglycols are suitable carriers for parenteral solutions. Solutions forparenteral administration contain preferably a water soluble salt of theactive ingredient, suitable stabilizing agents and, if necessary, buffersubstances. Antioxidizing agents such as sodium bisulfate, sodiumsulfite or ascorbic acid, either alone or combined, are suitablestabilizing agents. Also used are citric acid and its salts and sodiumethylenediaminetetraacetic acid (EDTA). In addition, parenteralsolutions can contain preservatives such as benzalkonium chloride,methyl- or propyl-paraben and chlorobutanol. Suitable pharmaceuticalcarriers are described in Remington's Pharmaceutical Sciences, astandard reference text in this field.

The compounds may also be formulated in rectal compositions such assuppositories or retention enemas, e.g., containing conventionalsuppository bases such as cocoa butter or other glycerides.

In addition to the formulations described previously, the compounds mayalso be formulated as a depot preparation. Such long acting formulationsmay be administered by implantation (for example subcutaneously orintramuscularly) or by intramuscular injection. Thus, for example, thecompounds may be formulated with suitable polymeric or hydrophobicmaterials (for example as an emulsion in an acceptable oil) or ionexchange resins, or as sparingly soluble derivatives, for example, as asparingly soluble salt.

Additionally, standard pharmaceutical methods can be employed to controlthe duration of action. These are well known in the art and includecontrol release preparations and can include appropriate macromolecules,for example polymers, polyesters, polyamino acids, polyvinyl,pyrolidone, ethylenevinylacetate, methyl cellulose, carboxymethylcellulose or protamine sulfate. The concentration of macromolecules aswell as the methods of incorporation can be adjusted in order to controlrelease.

Additionally, the agent can be incorporated into particles of polymericmaterials such as polyesters, polyamino acids, hydrogels, poly (lacticacid) or ethylenevinylacetate copolymers. In addition to beingincorporated, these agents can also be used to trap the compound inmicrocapsules.

The compositions may, if desired, be presented in a pack or dispenserdevice which may contain one or more unit dosage forms containing theactive ingredient. The pack may for example comprise metal or plasticfoil, such as a blister pack. The pack or dispenser device may beaccompanied by instructions for administration.

Useful pharmaceutical dosage forms, for administration of the compoundsof this invention can be illustrated as follows:

-   -   Capsules: Capsules are prepared by filling standard two-piece        hard gelatin capsulates each with the desired amount of powdered        active ingredient, 175 milligrams of lactose, 24 milligrams of        talc and 6 milligrams magnesium stearate.        -   Soft Gelatin Capsules: A mixture of active ingredient in            soybean oil is prepared and injected by means of a positive            displacement pump into gelatin to form soft gelatin capsules            containing the desired amount of the active ingredient. The            capsules are then washed and dried.    -   Tablets: Tablets are prepared by conventional procedures so that        the dosage unit is the desired amount of active ingredient. 0.2        milligrams of colloidal silicon dioxide, 5 milligrams of        magnesium stearate, 275 milligrams of microcrystalline        cellulose, 11 milligrams of cornstarch and 98.8 milligrams of        lactose. Appropriate coatings may be applied to increase        palatability or to delay absorption.    -   Injectable: A parenteral composition suitable for administration        by injection is prepared by stirring 1.5% by weight of active        ingredients in 10% by volume propylene glycol and water. The        solution is made isotonic with sodium chloride and sterilized.    -   Suspension: An aqueous suspension is prepared for oral        administration so that each 5 millimeters contain 100 milligrams        of finely divided active ingredient, 200 milligrams of sodium        carboxymethyl cellulose, 5 milligrams of sodium benzoate, 1.0        grams of sorbitol solution U.S.P. and 0.025 millimeters of        vanillin.

Gene Therapy Administration: Where appropriate, the gene therapy vectorscan be formulated into preparations in solid, semisolid, liquid orgaseous forms such as tablets, capsules, powders, granules, ointments,solutions, suppositories, injections, inhalants, and aerosols, in theusual ways for their respective route of administration. Means known inthe art can be utilized to prevent release and absorption of thecomposition until it reaches the target organ or to ensure timed-releaseof the composition. A pharmaceutically acceptable form should beemployed which does not ineffectuate the compositions of the presentinvention. In pharmaceutical dosage forms, the compositions can be usedalone or in appropriate association, as well as in combination, withother pharmaceutically active compounds.

Accordingly, the pharmaceutical composition of the present invention maybe delivered via various routes and to various sites in an animal bodyto achieve a particular effect (see, e.g., Rosenfeld et al. (1991),supra; Rosenfeld et al., Clin. Res., 3 9(2), 31 1A (1991 a); Jaffe etal., supra; Berkner, supra). One skilled in the art will recognize thatalthough more than one route can be used for administration, aparticular route can provide a more immediate and more effectivereaction than another route. Local or systemic delivery can beaccomplished by administration comprising application or instillation ofthe formulation into body cavities, inhalation or insulation of anaerosol, or by parenteral introduction, comprising intramuscular,intravenous, peritoneal, subcutaneous, intradermal, as well as topicaladministration.

The compositions of the present invention can be provided in unit dosageform wherein each dosage unit, e.g., a teaspoonful, tablet, solution, orsuppository, contains a predetermined amount of the composition, aloneor in appropriate combination with other active agents. The term “unitdosage form” as used herein refers to physically discrete units suitableas unitary dosages for human and animal subjects, each unit containing apredetermined quantity of the compositions of the present invention,alone or in combination with other active agents, calculated in anamount sufficient to produce the desired effect, in association with apharmaceutically acceptable diluent, carrier, or vehicle, whereappropriate. The specifications for the unit dosage forms of the presentinvention depend on the particular effect to be achieved and theparticular pharmacodynamics associated with the pharmaceuticalcomposition in the particular host.

Accordingly, the present invention also provides a method oftransferring a therapeutic gene to a host, which comprises administeringthe vector of the present invention, preferably as part of acomposition, using any of the aforementioned routes of administration oralternative routes known to those skilled in the art and appropriate fora particular application. The “effective amount” of the composition issuch as to produce the desired effect in a host which can be monitoredusing several end-points known to those skilled in the art. Effectivegene transfer of a vector to a host cell in accordance with the presentinvention to a host cell can be monitored in terms of a therapeuticeffect (e.g. alleviation of some symptom associated with the particulardisease being treated) or, further, by evidence of the transferred geneor expression of the gene within the host (e.g., using the polymerasechain reaction in conjunction with sequencing, Northern or Southernhybridizations, or transcription assays to detect the nucleic acid inhost cells, or using immunoblot analysis, antibody-mediated detection,mRNA or protein half-life studies, or particularized assays to detectprotein or polypeptide encoded by the transferred nucleic acid, orimpacted in level or function due to such transfer).

These methods described herein are by no means all-inclusive, andfurther methods to suit the specific application will be apparent to theordinary skilled artisan. Moreover, the effective amount of thecompositions can be further approximated through analogy to compoundsknown to exert the desired effect.

Furthermore, the actual dose and schedule can vary depending on whetherthe compositions are administered in combination with otherpharmaceutical compositions, or depending on individual differences inpharmacokinetics, drug disposition, and metabolism. Similarly, amountscan vary in in vitro applications depending on the particular cell lineutilized (e.g., based on the number of adenoviral receptors present onthe cell surface, or the ability of the particular vector employed forgene transfer to replicate in that cell line). Furthermore, the amountof vector to be added per cell will likely vary with the length andstability of the therapeutic gene inserted in the vector, as well asalso the nature of the sequence, and is particularly a parameter whichneeds to be determined empirically, and can be altered due to factorsnot inherent to the methods of the present invention (for instance, thecost associated with synthesis). One skilled in the art can easily makeany necessary adjustments in accordance with the exigencies of theparticular situation.

The following examples are offered by way of example, and are notintended to limit the scope of the invention in any manner.

EXAMPLES Identification and Cloning of Endostatin Genes

In the Example presented in this section, studies are described thatidentify novel canine genes, referred to herein as endostatin, which areinvolved in angiogenesis-related disorders, e.g., cancer.

Materials And Methods

1. Isolation of RNA from dog liver tissue. 50 mg of dog liver tissue wasdisrupted and homogenized by rotor-stator homogenizer (Kontes, Vineland,N.J.) and total RNA was purified using Rneasy Mini Kit followinginstructions from manufacturer (Qiagen Inc., Santa Clarita, Calif.). 30μg of RNA was isolated and was suspended in H20 at 0.5 μg/μl.

2. RT-PCR amplification and cloning of a region of canine collagen XVIIIencompassing endostatin. Endostatin is a C-terminal fragment of collagenXVIII (amino acid H1132-K1315 in murine collagen XVIII). A pair ofprimers were designed to amplify a region of canine collagen XVIII cDNAbased on consensus sequences from human (Accession No. L22548), mouse(Accession No. U03714) and chicken (Accession No. AF083440). The 5′primer: CCCTGGCGGGCAGATGACATCCTGGCC (SEQ ID NO:5) corresponding tonucleotide #766-792 of murine partial collagen XVIII cDNA the 3′ primer:CTCTTTGGCTTCCTTTTATTTCTTGAGGATTACAT (SEQ ID NO:6), corresponding tonucleotide #1569-1603 of murine partial collagen XVIII cDNA were usedfor the amplification reaction. The RT-PCR reaction was performed usingTitan One Tube RT-PCR Kit from Boehringer Mannheim GmbH (Germany). 0.5ug of dog liver RNA was denatured at 68° C. for 2 minutes. The reversetranscription reaction was performed at 50° C. for 30 minutes, and thePCR program was: hold at 94° C. for 3 minutes, followed by 35 cycles of30 seconds at 94° C., 30 seconds at 55° C., 1.5 minutes at 68° C. and 5minutes of extension at 68° C. The PCR products were analyzed byelectrophoresis.

The PCR products were cloned into Eukaryotic TA cloning vector pCR 3.1(Invitrogen, Carlsbad, Calif.) following manufacturer's instructions anddesignated as pCR3. 1-pro-ca-endostatin (pCR3.1 E:UC25432), depositedwith the American Type Culture Collection (ATCC), Manassas, Va.20110-2209, USA under Patent Deposit Designation PTA 2096. Threeindependent clones containing inserts of the expected size (0.8 kb) weresequenced (Advanced Genetic Analysis Center, St. Paul, Minn.). Thesequences were assembled and analyzed using DNAStar (DNAStar Inc.Madison, Wis.).

3. Subcloning of HA-tagged canine endostatin. The exact fragment ofcanine collagen XVIII corresponding to endostatin was subcloned intopDisplay vector by RT-PCR amplification of dog liver RNA using primers:5′ primer-CTAGAGATCTCACACCCACCAGGACTTCCAGC, 3′primer-CGTAGTCGACCTACTTGGAGAAGGAGGTCATGAC. To facilitate cloning, tworestriction enzyme sites Bgl II (5′ primer) and Sal I (3′ primer) wereincorporated into the primer sequences as shown by underline. The insertwas fused in-frame to the signal peptide and HA epitope sequencespresent in the vector. The stop codon TAG (shown in bold) of endostatinwas included in the 3′ primer to terminate translation, therefore thevector-encoded PDGFR transmembrane domain downstream of the insert wouldnot be translated in the final plasmid construct,pDisplay-HA-ca-endostatin (PdisplayE:UC25433), deposited with the ATCCunder Patent Deposit Designation PTA-2097.

4. Subcloning of canine endostatin (without HA tag). Canine endostatinwas subcloned into pSecTag2 B vector by RT-PCR amplification of dogliver RNA using primers: 5′ primer-GATTAAGCTTCACACCCACCAGGACTTCCAGCT(SEQ ID NO:7), 3′ primer-CTGAGAATTCCTACTTGGAGMGGAGGTCATGAC (SEQ IDNO:8). To facilitate cloning, two restriction enzyme sites Hind III (5′primer) and EcoR I (3′ primer) were incorporated into the primersequences as shown by underline. The insert was fused in-frame to thesignal peptide sequences present in the vector. The stop codon TAG(shown in bold) of endostatin was included in the 3′ primer to terminatetranslation. The final plasmid construct was designatedpSecTag2-ca-endostatin (pSecTag2E:UC 25434) deposited with the ATCCunder Patent Deposit Designation PTA-2098.

5. Cloning of murine endostatin. In order to compare theanti-angiogenesis activity of cloned canine endostatin to that of itsmurine counterpart, cDNAs encoding murine endostatin were RT-PCRamplified from mouse liver RNA and cloned into pDisplay vector. Theprimers used for amplifying murine endostatin were:5′-CTAGAGATCTCATACTCATCAGGACTTTCAGC (SEQ ID NO:9),3′-GCTAGTCGACCTATTTGGAGAAAGAGGTCATG (SEQ ID NO:10). The flankingrestriction sites Bgl II (5′ primer) and Sal I (3′ primer) areunderlined and the stop codon is shown in bold. The resultant plasmidwas designated as pDisplay-HA-mu-endostatin (Accession No. U03714).

Results

The cDNAs encoding a fragment of canine collagen XVIII which containsthe coding region of endostatin were amplified by RT-PCR from dog livermRNA using primers designed from consensus sequences from severalspecies. The nucleotide sequence of pro-endostatin is shown in FIG. 2(SEQ ID NO:1), and the predicted amino acid sequence is shown in FIG. 3(SEQ ID NO:2). The region corresponding to endostatin based on homologyis in bold in FIG. 3 and the exact nucleotide sequence and predicatedamino acid sequence of canine endostatin (184 amino acids) is shown inFIG. 4 (SEQ ID NO:3) and FIG. 5 (SEQ ID NO:4) respectively. FIG. 6 showsthe alignment of all known amino acid sequences of endostatin, and thedegree of homology between canine endostatin and that of human, mouseand chicken is 84%, 83% and 76% respectively.

Expression of Endostatin Genes

In the Example presented in this section, studies are described thatidentify methods to express and assay the novel canine endostatin genes.

Materials And Methods

1. Transfection of endostatin. Human 293 cells grown in 6 well platewere transfected with 2.5 ug of plasmids encoding canine or murineendostatin using CalPhos Mammalian Transfection Kit (CLONTECHLaboratories, Inc., Palo Alto, Calif.).

2. Detection of endostatin by immunofluorescence. 2 dayspost-transfection, cells were fixed in 4% paraformaldehyde, permeablizedin 0.05% Triton X100, and blocked in 1% goat serum (all chemicals fromSigma, St. Louis, Miss.). HA.11 (BabCO, Richmond, Calif.), a monoclonalantibody against HA epitope was used at 1:500 dilution to stain thecells, and TR1TC conjugated anti-mouse IgG (Sigma) was used at 1:1,000for detection. The immunofluorescent cells were visualized under NikonTE 300 microscope.

3. Detection of endostatin by immunoblot analysis. 2 dayspost-transfection, cells were harvested by lysis in Tris-Glycine SDSSample Buffer (NOVEX, San Diego, Calif.). The culture supernatants wereharvested by centrifugation at 3000 rpm for 15 minutes. The proteinswere separated by 4-20% SDS-PAGE and transferred to PVDF membrane(NOVEX, San Diego, Calif.). For immunoblot analysis, HA antibody againstHA epitope were diluted 1:500 and incubated with the blot for 1.5 hours.After incubating for 30 minutes with alkaline phosphatase conjugatedanti-mouse IgG (1:10,00, Boehringer Mannheim, Indianapolis, Ind.), thebound antibody was detected using phosphatase substrate BCIP/NBT (KPL,Gaithersburg, Md.).

4. Endothelial cell proliferation assay. The anti-proliferative effectof cloned canine endostatin was tested using bovine pulmonary arteryendothelial cells (C-PAE, ATCC, Manassas, Va.). The cells (104cells/well) were plated in 24-well collagen I-coated plates(Collaborative Biomedical Products, Bedford, Mass.) in OptiMEM (GIBCOBRL, Rockville, Md.) with 2% fetal bovine serum (Nova-Tech Inc., GrandIsland, Nev.).

5. After 24 hour incubation, the medium was replaced with conditionedmedium from transfected 293 cells supplemented with 1 ng/ml bFGF(Collaborative Biomedical Products, Bedford, Mass.). The cells weretrypsinized 48 hours later and viable cells were counted using trypanblue staining and hemacytometer. The results were analyzed using Prizm2.01 (Graphpad Software Inc., San Diego, Calif.).

Results

Expression of canine endostatin. The cDNAs encoding canine endostatinwere subcloned into mammalian expression vector pDisplay. Becauseendostatin is a fragment of a secreted protein and normally circulatesin the blood, the proteins were fused, in-frame at the N-terminus to themurine 1 g k-chain leader sequence which directs the protein to thesecretory pathway, followed by fusion to hemagglutinin A epitope tag(HA) which allows for detection of expressed protein. Human 293 cellswere transfected with plasmids encoding signal sequence and HA-taggedendostatin from dog and mouse, the cells were harvested 48 hours posttransfection for analysis. FIG. 7 shows the results of immunofluorescentassay. The staining patterns of endostatin are characteristic ofperinuclear endoplasmic reticulum and trans-Golgi, consistent with thenotion that this protein is directed to the secretory pathway.

1. The expression of endostatin was further studied by immunoblotanalysis. The expression of transfected endostatin was detected fromboth cell lysates (FIG. 8, lane 2 and 4) and culture supernatants (FIG.8, lane 6 and 8). Several forms of intracellular endostatin withmolecular weights ranging 20-25 kDa (FIG. 8, lane 2 and 4) exist. Theseare likely intermediates of protein maturation since only one discreteband was seen in the secreted form.

2. Inhibition of endothelial cell proliferation. One unique feature ofendostatin is its ability to specifically inhibit endothelial cellproliferation (0□Reilly et al., 1997, Cell 88(2):277-85; O□Reilly etal., 1994, Cell 79(2):3 15-28). Bovine pulmonary artery endothelialcells (CPAE cells) (Dhanabal et al., 1999) were stimulated with basicfibroblast growth factor (bFGF) in the presence or absence of endostatinproteins produced from conditioned media from transfected 293 cells. Theproliferation rate was normalized against control cells which weretreated with bFGF and conditioned media from green fluorescent protein(GFP) transfected cells. The results from four independent experimentswere summarized in FIG. 9. CPAE cells proliferated slowly without bFGFactivation (38% of that of control). The addition of HA-tagged canineendostatin inhibited the stimulating effect of bFGF with CPAE cellsproliferating at 53% of that of control respectively. The inhibitoryactivity seen with HA-tagged canine endostatin was comparable to thatwith the murine proteins (both at 57% level of controls). Thedifferences in proliferation rate between control and each treated groupwere all statistically significant (P<0.05). In contrast, treatment withendostatin did not inhibit proliferation of the epithelial 293 cells,suggesting that the inhibitory effect of endostatin is specific toendothelial cells. Similar results were obtained using untagged canineendostatin (FIG. 10). The addition of canine endostatin specificallyinhibited the stimulating effect of bFGF with CPAE cells proliferatingat 59% of that of control. The differences in the proliferation ratebetween control and each treated group were all statisticallysignificant (P<0.005, n=3).

Discussion

Here the cloning of the canine angiogenesis inhibitor endostatin isreported. It shares approximately 80% homology with its human and murinecounterparts. To facilitate secretion of cloned proteins, a signalsequence from mouse 1 g k-chain was fused to the N-terminus ofendostatin. Immunofluorescent studies and immunoblot assays confirmedthat the proteins were localized to the secretory pathway and secretedinto conditioned media. Canine endostatin was also shown to specificallyinhibit endothelial cell proliferation at a level comparable to itsmurine counterpart.

Mouse endostatin has been shown to inhibit the growth of a wide varietyof primary and metastatic tumors. Furthermore, the treatment withendostatin can be repeated many times without inducing drug resistanceor side effects. Since these angiogenesis inhibitors are directed at anovel target (endothelial cells), they can also be conveniently combinedwith other cancer therapies such as surgery, chemotherapy, radiationtherapy and immunotherapy to achieve superior therapeutic effects. Theseproperties have made endostatin a very attractive candidate for treatingcanine cancers, where a safe, efficacious, and broad-spectrum therapy isvery much in need. However, the successful application of endostatin asa cancer therapy probably will involve repeated, continuing treatment inorder to achieve long-term tumor suppression and dormancy (Boehm et al.,1997, Nature 390(6658):404-407). The cloning and identification ofcanine endostatin allows for the treatment of dog tumors usingspecie-specific angiogenesis inhibitors, thereby minimizing the risk ofevoking immune responses under repeated administration. Finally,spontaneous canine tumors are very similar to their human correlates inhistopathologic and biologic behavior (MacEwen, 1990, Cancer MetastasisRev 9(2): 125-36), therefore experimental results obtained from caninetumors will also provide valuable information for human cancer biologyand treatment.

The present invention is not to be limited in scope by the specificembodiments described herein, which are intended as single illustrationsof individual aspects of the invention, and functionally equivalentmethods and components are within the scope of the invention. Indeed,various modifications of the invention, in addition to those shown anddescribed herein will become apparent to those skilled in the art fromthe foregoing description and accompanying drawings. Such modificationsare intended to fall within the scope of the appended claims.

All publications and patent applications mentioned in this specificationare herein incorporated by reference to the same extent as if eachindividual publication or patent application was specifically andindividually indicated to be incorporated by reference.

1-11. (Cancelled)
 12. A transgenic, non-human animal which has beengenetically engineered to contain a transgene comprising a nucleic acidselected from the group consisting of SEQ ID NO: 1, SEQ ID NO: 3, anisolated nucleic acid molecule that encodes an endostatin consisting ofSEQ ID NO: 2, and an isolated nucleic acid molecule that encodes andendostatin consisting of SEQ ID NO:
 4. 13. The transgenic, non-humananimal of claim 12, wherein the transgene is expressed.
 14. An isolatedpolypeptide comprising an amino acid sequence of: a) SEQ ID NO: 2; or b)SEQ ID NO:
 4. 15. The antibody which binds to the isolated polypeptideof claim
 14. 16. An isolated polypeptide comprising an amino acidsequence encoded by an isolated nucleic acid molecule, which hybridizesunder highly stringent conditions to the complement of a nucleic acidselected from the group consisting of SEQ ID NO: 1, SEQ ID NO: 3, anisolated nucleic acid molecule that encodes an endostatin consisting ofSEQ ID NO: 2, and an isolated nucleic acid molecule that encodes andendostatin consisting of SEQ ID NO:
 4. 17. An isolated polypeptidecomprising an amino acid sequence encoded by an isolated nucleic acidmolecule, which hybridizes under moderately stringent conditions to thecomplement of a nucleic acid selected from the group consisting of SEQID NO: 1, SEQ ID NO: 3, an isolated nucleic acid molecule that encodesan endostatin consisting of SEQ ID NO: 2, and an isolated nucleic acidmolecule that encodes and endostatin consisting of SEQ ID NO:
 4. 18. Anisolated fusion polypeptide comprising a fusion peptide and an aminoacid sequence of: a) SEQ ID NO: 2; or b) SEQ ID NO:
 4. 19. An isolatedfusion polypeptide comprising a fusion peptide and an amino acidsequence encoded by an isolated nucleic acid molecule, which hybridizesunder highly stringent conditions to the complement of a nucleic acidselected from the group consisting of SEQ ID NO: 1, SEQ ID NO: 3, anisolated nucleic acid molecule that encodes an endostatin consisting ofSEQ ID NO: 2, and an isolated nucleic acid molecule that encodes andendostatin consisting of SEQ ID NO:
 4. 20. An isolated fusionpolypeptide comprising a fusion peptide and an amino acid sequenceencoded by an isolated nucleic acid molecule, which hybridizes undermoderately stringent conditions to the complement of a nucleic acidselected from the group consisting of SEQ ID NO: 1, SEQ ID NO: 3, anisolated nucleic acid molecule that encodes an endostatin consisting ofSEQ ID NO: 2, and an isolated nucleic acid molecule that encodes andendostatin consisting of SEQ ID NO:
 4. 21. A method for treating anangiogenesis-related disorder in a subject comprising administering tothe subject a compound which modulates the function, activity and/orexpression of an endostatin sequence in the subject.
 22. The method ofclaim 21, wherein the compound enhances or increases the function,activity and/or expression of the endostatin sequence.
 23. The methodsof any one of claims 21-22, wherein the compound is selected from thegroup consisting of a small organic molecule, an antibody, a ribozyme oran antisense molecule.
 24. The method of any one of claims 21-22,wherein the angiogenesis-related disorder is selected from the groupconsisting of cancer; angiogenesis-dependent cancer, comprising solidtumors, blood born tumors such as leukemias, and tumor metastases;benign tumors, comprising hemangiomas, acoustic neuromas, neurofibromas,trachomas, and pyogenic granulomas; rheumatoid arthritis; psoriasis;ocular angiogenic diseases, comprising diabetic retinopathy, retinopathyof prematurity, macular degeneration, corneal graft rejection,neovascular glaucoma, retrolental fibroplasia, rubeosis; Osler-WebberSyndrome; myocardial angiogenesis; plaque neovascularization;telangiectasia; hemophiliac joints; angiofibroma; wound granulation;corornary collaterals; cerebral collaterals; arteriovenousmalformations; ischemic limb angiogenesis; diabetic neovascularization;macular degeneration; fractures; vasculogenesis; hematopoiesis;ovulation; menstruation; and placentation.
 25. The method of claim 21,wherein the endostatin sequence encodes an amino acid sequencecomprising: a) SEQ ID NO: 2; or b) SEQ ID NO:
 4. 26. The method of claim21, wherein the subject is a dog.
 27. A method for identifying acompound which modulates expression of an endostatin sequencecomprising: a) contacting a test compound to a cell that expresses anendostatin sequence; b) measuring a level of endostatin sequenceexpression in the cell; c) comparing the level of endostatin sequenceexpression in the cell in the presence of the test compound to a levelof endostatin sequence expression in the cell in the absence of the testcompound, wherein if the level of endostatin sequence expression in thecell in the presence of the test compound differs from the level ofexpression of the endostatin sequence in the cell in the absence of thetest compound, a compound that modulates expression of an endostatinsequence is identified.
 28. The method of claim 27, wherein theendostatin sequence is endogenously expressed within the cell.
 29. Themethod of claim 27, wherein the endostatin sequence encodes an aminoacid sequence comprising: a) SEQ ID NO: 2; or b) SEQ ID NO:
 4. 30. Themethod of claim 27, wherein the endostatin sequence comprises: a) anucleic acid as shown in SEQ ID NO: 1; or b) a nucleic acid as shown inSEQ ID NO:
 3. 31. A method for identifying a compound which modulatesactivity of an endostatin sequence product comprising: a) contacting atest compound to a cell that expresses an endostatin sequence product;b) measuring a level of endostatin sequence product in the cell; c)comparing the level of endostatin sequence product activity in the cellin the presence of the test compound to a level of endostatin sequenceproduct activity in the cell in the absence of the test compound,wherein if the level of endostatin sequence product activity in the cellin the presence of the test compound differs from the level ofendostatin sequence product activity in the cell in the absence of thetest compound, a compound that modulates activity of an endostatinsequence product is identified.
 32. The method of claim 31, wherein theendostatin sequence product comprises: a) SEQ ID NO: 2; or b) SEQ ID NO:4.
 33. A method for modulating the activity and/or expression of anendostatin sequence in a cell comprising administering to the cell acompound which modulates the activity and/or expression of an endostatinsequence in the cell.
 34. The method of claim 33, wherein the compoundis selected from the group consisting of a small organic molecule, anantibody, a ribozyme or an antisense molecule.
 35. The method of claim33, wherein the endostatin sequence encodes an amino acid sequencecomprising: a) SEQ ID NO: 2; or b) SEQ ID NO:
 4. 36. The method of claim33, wherein the endostatin sequence comprises: a) a nucleic acid asshown in SEQ ID NO: 1; or b) a nucleic acid as shown in SEQ ID NO: 3.